In vitro flowering of Fortunella hindsii (Champ.)

Branch internodes of mature plants and stem internodes of seedlings of Fortunella hindsii flowered in vitro on half-strength MT (Murashige and Tucker 1969) basal medium supplemented with benzyladenine, adenine, 6---dimethylallylaminopurine and kinetin. The highest percentage of flowering was achieved with explants originating from branch internodes of flowering plants close to the apex on half-strength MT basal medium containing 5% sucrose and 0.01 mg 1^–1 BA in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 4-day exposure to BA, but shoot formation could be initiated even without exposure to BA. First branch internode segments on MT basal medium containing 5% sucrose were prolific in flower (85%) production. The sucrose treatment affected the flower bud size distribution. There were about 13 flower buds per culture in the largest size category (>5 mm).     

Somatic embryogenesis and plant regeneration from fragments of immature inflorescences and coleoptiles of durum wheat

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchid through formation of protocorm-like bodies

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.     

Somatic embryogenesis of Agave victoria-reginae Moore

Plant regeneration through direct somatic embryogenesis of leaf blade explants from in vitro propagated plants of Agave victoria-reginae Moore, is described. Somatic embryogenesis was evident in a 6-week period on agarsolidified MS medium supplemented with L2 vitamins and 2,4-dichlorophenoxyacetic acid (1,4 µM), and germination of somatic embryos was achieved after 8 weeks on half-strength MS medium and 4 weeks on half-strength SH medium, both lacking growth regulators. Hyperhydricity of somatic embryos and plantlets was reduced by the use of vented culture vessel lids during the last 4 weeks on SH medium. Shoot proliferation was obtained, and hyperhydricity was eliminated on a modified MS medium (with NH₄NO₃ reduced to 5 mN) supplemented with kinetin (4.6 µM) and 1-naphthaleneacetic acid (1.6 µM) and the use of vented culture vessel lids.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog - LOG-1 MS modified medium by Castro-Concha et al. (1990) - L2 Phillips and Collins (1979) vitamins - SH Schenk and Hildebrandt     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

In vitro propagation of the endangered plant <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>: assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis

The effect of different cytokinins on multiple shoot regeneration from shoots of Centaurea ultreiae was studied. The culture system consisted of solid basal half-strength Murashige and Skoog medium supplemented with one of four cytokinins [6-benzyladenine (BA), zeatin, kinetin, or N⁶-(2-isopentyl) adenine (2-iP)] at each of five different concentrations. The highest multiplication rate (5.52 shoots per explant) was obtained in the medium supplemented with 4.44 μM BA. Shoots were successfully rooted (91% success) by dipping the basal end into a solution containing 10 M 1-naphthaleneacetic acid for 30 s. Genetic stability of the regenerated plants was assessed by random amplified polymorphic DNA (RAPD) analysis and flow cytometry. In the initial randomly selected plant material (control) and 20 of its regenerants, 2,688 bands were generated by RAPD with 12 different primers, and the same banding profiles were exhibited. Molecular and cytological analyses did not reveal genomic alterations in any of the regenerated plants obtained on medium containing 4.44 μM BA. The success of acclimatization to environmental conditions—100% of plants were successfully acclimatized—suggests that the micropropagation system described is a reliable method for propagation of C. ultreiae.     

Shoot initiation and multiplication from a mature tree of Terminalia arjuna roxb

Summary This study describes a protocol for the regeneration of complete plantlets of Terminalia arjuna from nodal explants of mature trees. Shoot multiplication from nodal explants was achieved by culturing on Murashige and Skoog (MS) medium containing different concentrations of 6-benzyladenine (BA), thidiazuron or kinetin, or BA in combination with &#x03B1;-naphthaleneacetic acid (NAA). The best shoot multiplication response was obtained from nodal explants grown on modified MS (half-strength major salts and Fe-EDTA) medium containing 4.44 &#x03BC;M BA and 0.53 &#x03BC;M NAA. Seasonal variations significantly affected the proliferation potential of nodal explants and best proliferation was observed from explants collected during April to May. Microshoots were rooted on half-strength MS medium with 4.92 &#x03BC;M IBA. The rooted shoots were acclimatized successfully.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

Efficient in vitro germination and shoot proliferation of chilling-treated water chestnut (Trapa japonica Flerov) embryonal explants

Summary Embryonal explants from water chestnut (Trapa japonica Flerov) seeds germinated with high efficiency following a 40-d cold treatment at 5°C on half-strength MS (Murashige and Skoog) medium supplemented with 2.7 μM N⁶-benzyladenine (BA), 0.5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM gibberellic acid (GA₃). Control and chill-treated (different durations) embryonal explants were cultured onto media which contained half-strength MS medium supplemented with different concentrations and combinations of cytokinins [BA, thidiazuron (TDZ), kinetin, zeatin], auxin (NAA) and GA₃. A liquid half-strength MS medium with 1.1 μM BA and 0.5 μM NAA resulted in the best shoot proliferation of control or chill-treated explants, and the addition of 0.5 μM GA₃ stimulated axillary shoot elongation. Germination and shoot proliferation were always greater for chill-treated explants compared with control explants under the same culture conditions. Shoots produced in vitro rooted 100% of the time in a liquid half-strength MS medium with 1.1 μM BA, 0.5 μM NAA and 1.1 μM indole-3-butyric acid, and the regenerated plantlets were established successfully in a water chestnut paddy field.     

Direct shoot regeneration from lamina explants of two commercial cut flower cultivars of Anthurium andraeanum Hort.

Summary Direct plant regeneration from flowering plant-derived lamina explants of Anthurium andraeanum Hort. cultivars Tinora Red and Senator was established on modified Murashige and Skoog (MS) medium. Cultivar difference, stage of source lamina and the position of explant in lamina, medium pH, and type of growth regulators significantly influenced direct plant regeneration. Explants from young brown lamina were superior to young green lamina. The half-strength MS medium containing 1.11 μM N⁶-benzyladenine (BA), 1.14 μM indole-3-acetic acid, and 0.46 μM kinetin at pH 5.5 was most effective for induction of shoot formation. Explants from the proximal end of the source lamina gave rise to a higher number of shoots compared to the mid and distal regions. Cultivar Tinora Red was more regenerative than Senator in terms of number of shoots per explant. The use of a lower BA concentration (0.44 μM) was essential for callus-free shoot multiplication during subculture. Regenerated shoots could be induced to form roots on half-strength MS medium supplemented with 0.54 μM α-naphthaleneacetic acid and 0.93 μM kinetin. More than 300 plantlets of each eultivar were harvested from a single source lamina within 200 d of culture. Most plantlets (95%) survived after acclimation in soil.     

In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi-an endangered medicinal plant native to Taiwan

Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l⁻¹ BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shool multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l⁻¹ indole-3-butyric acid and 0.1–0.2 mg l⁻¹ α-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2+DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4wk) and ex vitro-acclimated plantlets.     

Effect of auxins on indirect <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis and expression of <Emphasis Type="Italic">gusA</Emphasis> transgene in a lectinaceous medicinal plant, <Emphasis Type="Italic">Euphorbia nivulia</Emphasis> Buch.-Ham.

Summary The types of auxin in Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Effect of growth regulators on rapid micropropagation and psoralen production in <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

A rapid and efficient micropropagation system was developed for Psoralea corylifolia, an endangered, valuable medicinal plant. Multiple shoot buds were obtained in half-strength liquid Phillips–Collins (L2) medium supplemented with 5 μM benzylaminopurine (BA) and 5 μM thidiazuron (TDZ) from apical bud explants of 1-week-old cultures. The shoot buds were subcultured on enriched solid L2 medium supplemented with different concentrations and combinations of BA, kinetin (KIN), 2-isopentenyladenine (2iP), TDZ, bavistin (BVN) and trimethoprim (TMP). Enriched solid L2 medium supplemented with 2 μM BA, 1 μM TDZ and 100 mg l⁻¹ BVN were more effective in producing greater number of shoots per explant (85.2 ± 0.9 shoots/explant) after 4 weeks of culture. The regenerated shoots (40–50 mm in length) rooted and accompanied by hardening upon transfer to 50 μM indole-3-butyric acid (IBA) for 15 min and followed by planting in sterile soil mixture and vermiculate (3:1 v/v), with 50 ml of one-eight strength L2 basal salt solution devoid of sucrose and inositol, supplemented with 5 μM IBA and 100 mg l⁻¹ BVN. The plants achieved 100% rooting with hardening. Subsequently the rooted plants were successfully established in the field. The survival percentage differed with seasonal variations. The concentration of psoralen was evaluated in different tissues of ex vitro and in vivo grown plants by high-performance liquid chromatography (HPLC). Psoralen content was increased in leaves (2.97%), roots (2.38%), stems (5.40%) and seeds (1.63%) of ex vitro plants than the in vivo plants. This system facilitates for commercial and rapid propagation of P. corylifolia for conservation strategies and phytomedicine production.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

An efficient plant regeneration system for Mucuna pruriens L. (DC.) using cotyledonary node explants

Summary The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.