Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

An improved method of in vitro propagation ofDioscorea bulbifera

Nodal stem segments ofDioscorea bulbifera were induced to form plantlets in vitro. Rooted plantlets were obtained on Murashige-Skoog [14] revised medium supplemented initially with 5 mg 1⁻¹ kinetin and subsequently with 5 mg l⁻¹ indole-butyric acid. By increasing the kinetin concentration from 5 mg l⁻¹ to 10 mg l⁻¹, the number of shoots forming per node was increased from five to eight. When kinetin was substituted with 6-benzylaminopurine at only 1 mg l⁻¹, nine shoots formed on each node. Each shoot could be excised from the node and induced to form a new crop of multiple shoots. Rooted plantlets could be successfully transferred to in vivo conditions.     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Micropropagation studies inCeropegia SPP

Summary The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l⁻¹) N⁶-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l⁻¹) BA and 23.2 μM (5 mg·l⁻¹) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l⁻¹) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l⁻¹) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA. Rooting of the shoots was possible both byin vitro andex vitro means.     

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.     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

Cytokinins,auxins and activated charcoal affect organogenesis and anatomical characteristics of shoot-tip cultures of lisianthus [<Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> (RAF.) Shinn]

Summary Variants from seed-propagated Lisianthus [Eustoma grandiflorum (Raf.) Shinn] were shoot-tip cultured to observe the effects of cytokinins, auxins and activated charcoal on organogenesis and anatomical characteristics. N⁶-Benzyladenine (BA) and kinetin at high concentrations (13.32–22.2 and 13.94–23.23 μM) resulted in good shoot formation but high percentages of hyperhydric shoots. Increased indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) concentrations favored root formation, while increased naphthaleneacetic acid concentration adversely affected root formation. Both shoot and root development were suppressed by activated charcoal. The highest percentage of regeneration and the largest number of glaucous shoots with an average of 15 shoots per explant after 4 wk of culture were obtained when the shoot tips were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 4.44 μM BA and 1.47–4.92 μMIAA and IBA. In vitro-grown leaves had a higher number of stomata than field-grown leaves but the length and diameter of stomata showed no significant difference between the two types. Field-grown leaves had well-developed epicuticular wax layers. which were not observed on hyperhydric leaves. Hyperthydric plantlets could not survive when transplanted to soil, whereas glaucous plantlets survived in more than 80% of cases. Variation in soil type resulted in a slight difference in plantlet survival. Based on the results of our experiment, this protocol should be useful for the rapid micropropagation of lisianthus.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

In vitro flower induction in roses

Summary Vegetatively propagated plantlets of six rose cultivars were induced to flower in vitro on media containing full-strength Murashige and Skoog (MS) inorganic salts, Gamborg's B5 organic elements with 400 mg l⁻¹ myo-inositol, and different phytohormone combinations of 6-benzyladenine (BA) with α-naphthaleneacetic acid (NAA); thidiazuron (TDZ) with NAA; and zeatin (ZT) with NAA. The most efficient flower bud induction (49.1% and 44.1%) was obtained on media supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.1 mg l⁻¹ (0.54 μM) NAA or 0.5 mg l⁻¹ (2.28 μM) ZT and 0.1 mg l⁻¹ (0.54 μM) NAA for cultivar Orange Parade. Scanning electron microscopy (SEM) showed that in vitro flower bud induction occurred mostly between 15 and 30 d in induction medium through the normal flower development processes. With TDZ and ZT as the best choice for flower induction in all six cultivars tested, different rose cultivars varied in their responses to phytohormone treatments. Our study also revealed that the total time from original culture and subculture time before flower induction were two very important factors for in vitro flower induction. Plantlets 156–561 d from original culture and subcultured for 45 d were the best for flower induction. These authors contributed equally to this work.     

Humid incubation period and plantlet age influence acclimatization and establishment of micropropagated grapes

Summary In vitro rooted grape (Vitis vinifera L.) plantlets (4 or 8 wk from culturing microcuttings) were plantedex vitro in polythene sachets (24×12 cm) filled to one-third their height with planting mixture. The sachets were misted, closed, and incubated at ambient temperature (25–30°C) under 16 h photoperiod (40–50 μ E·m^−2.·sec⁻¹) for 1,2, or 3 wk before opening. Maximum establishment with no or minimum damage toin vitro formed leaves was obtained with 3 wk of closed sachet incubation in both age groups. Opening the sachet at 2 wk from planting resulted in marginal scorching of lower leaves and some reduction in establishment and vigor. Opening at 1 wk led to severe leaf scorching and significant reduction in establisment particularly in 4-wk-oldin vitro plantlets while growth was more affected in 8-wk-old ones. Relative humidity ofin vitro culture vessel was 68–75% while closed sachets had RH values of 45–77% depending on length of incubation and ambient RH. Diurnal variation in RH of sachet in relation to ambient RH was the major factor that facilitated acclimatization rather than the overall fall in RH during the period of closed incubation. Satisfactory acclimatization of plantlets to withstand the open sachet RH (50–55%) by 3 wk and the ambient RH (30–40%) by 4 wk was achieved. Monitoring water loss from detached leaves of plantlets showed a significant reduction between the date of planting and Week 3, and again between Weeks 3 and 4. Comparison of growthin vitro andex vitro suggested that shifting toex vitro earlier was more beneficial. This observation was confirmed by transferring 3-, 4-, and 5-wk-old plantlets fromin vitro rooting medium toex vitro and recording the growth at 8 wk fromin vitro culturing when 3 wkin vitro plus 5 wkex vitro combination showed maximum vigor. The leftover stumps after subculturing of 1–4-mo.-old stock cultures could also be effectively used forex vitro establishment.     

Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

An improved system for the <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Thapsia garganica</Emphasis> via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l⁻¹ added to Murashige and Skoog (MS) medium with 30 g l⁻¹ sucrose and 8 g l⁻¹ agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l⁻¹ kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l⁻¹ kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l⁻¹ kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l⁻¹ medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite^®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.     

A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. f. ex diels. axillary shoots in vitro

The effect of different concentrations and activities of cytokinins on the morphogenesis of regenerated Rhododendron forrestii Balf. f. ex Diels. shoots taken from nodal segments were tested. We evaluated zeatin, zeatin riboside, izopentyladenine, izopentyladenine riboside, kinetin, kinetin riboside, benzylaminopurine, benzylaminopurine riboside. The experimental results were evaluated by mathematical methods and regression analysis describing the effect of isoprenic and aromatic type of cytokinins. On the basis of this modelling, maximum axillary shoot production was attained with medium supplemented with 2.0 mg·l⁻¹ izopentyladenine riboside, 2.0 mg·l⁻¹ benzylaminopurine and 20 g·l⁻¹ sucrose. Minimal axillary shoots were produced with kinetin and kinetin riboside.     

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.