Use of protocorm-like bodies for studying alkaloid metabolism in <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

Prolific shoot regeneration of <Emphasis Type="Italic">Astragalus cariensis</Emphasis> Boiss

Prolific shoot regeneration via organogenesis was induced from leaf and leaf petiole explants of the endemic Astragalus cariensis species on Murashige and Skoog (MS) medium with α-naphthaleneacetic acid (NAA) and benzyladenine (BA) within 8 week. The highest number of shoots (23/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. Elongated shoots were successfully rooted in MS medium with 0.5 mg/l indole-3-butyric acid. Rooted plantlets were acclimatized in pots containing 1:1 mixture of peat and perlite.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.     

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

Rapid Multiplication and Induction of Early In Vitro Flowering in Dendrobium Primulinum Lindl

Dendrobium primulinum is an important epiphytic orchid. A successful protocol for mass multiplication and early in vitro flowering was developed. Immature embryos of 4 week after pollination exhibited about 96% germination within 30 days of culture on MS medium containing sucrose (3%) (w/v), NAA and BA (6 and 9 μM) in combination. Protocorm-like bodies (PLBs) formed from the germinating seeds on the germination medium. Rooted plantlets were formed within 2-3 wk on MS medium containing sucrose (3%), NAA and BA (3 and 12 μM in combination) where about 29 shoot/buds produced per cycle of 4 wk interval. The well rooted plantlets produced 4-5 floral buds per spike when they were maintained on MS medium containing sucrose (3%), fresh apple juice (10%) (v/v) for four wk followed by on MS medium freed of apple juice but enriched with NAA and BA (3 and 12 μM respectively). The hardened plantlets were transferred to community potting mix where the about 80% transplants survived after two months of transfer.     

Induction of somatic embryogenesis and organogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L., a dye-yielding medicinal plant

Oldenlandia umbellata L., commonly known as “Indian madder”, is an ancient Indian herb valued as a source of red color dye and various medicinal products. In this study, successful protocols have been developed for induction of somatic embryogenesis and organogenesis in O. umbellata. Emerging young leaves, shoot apices, and stems were used as explants, grown on Murashige and Skoog (MS) media supplemented with various auxins, including indole acetic acid, indole butyric acid, napthaleneacetic acid (NAA), and 2,4-Dichlorophenoxyacetic acid, each at levels ranging between 0.1 and 0.5 mg/l, cytokinins, including benzyladenine (BA) and kinetin, each at concentration ranging between 0.5 and 5 mg/l, with and without coconut milk (CM) at levels of 0.5–5%. For callus induction, NAA at 2.5 mg/l was optimal; while, for rapid embryogenic callus induction, 0.2 mg/l NAA, 0.5 mg/l BA, and 0.1% CM induced the highest frequency (95.86%). Shoots developed upon transfer of embryogenic calli to MS medium containing 1.5 mg/l BA, 0.3 mg/l NAA and 1% CM. For root induction, 0.3 mg/l NAA and 1.0% CM promoted highest and earliest rooting. C. Rajasekaran contributed equally to this work.     

Shoot organogenesis in elite clones of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

An efficient shoot organogenesis system has been developed from mature plants of selected elite clones of Eucalyptus tereticornis Sm. Cultures were established using nodal explants taken from freshly coppice shoots cultured on Murashige and Skoog medium containing 58 mM sucrose, 0.7% (w/v) agar (MS medium) and supplemented with 2.5 μM benzyladenine (BA) and 0.5 μM α-naphthaleneacetic acid (NAA). Shoot organogenesis was achieved from leaf segments taken from elongated microshoots on MS medium supplemented with 5.0 μM BA and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of cefotaxime to the medium promoted shoot differentiation, whereas carbenicillin and cephalexin inhibited shoot differentiation. Maximum shoot bud organogenesis (44.6%) occurred in explants cultured on MS medium supplemented with 5.0 μM BA, 1.0 μM 2,4-D and 500 mg/l cefotaxime. Leaf maturity influenced shoot regeneration, with maximum shoot organogeneisis (40.5%) occurring when the source of explants was the fifth leaf (14–16 days old) from the top of microshoot. Shoot organogenic potential also varied amongst the different clones of E. tereticornis. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses indicated clonal uniformity of the newly formed shoots/plants, and these were also found to be true-to-type.     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

Effects of different spectral lights on <Emphasis Type="Italic">Oncidium</Emphasis> PLBs induction,proliferation, and plant regeneration

The effects of different spectral light distribution on in vitro induction and proliferation of Oncidium protocorm-like bodies (PLBs) and subsequent growth of plantlets were investigated. Shoot tips (5 mm in length) of proliferating shoots of Oncidium “Gower Ramsey” were vertically incubated on 1/2 Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 6-benzyladenine (BA), and grown under either monochromatic red light-emitting diodes (LEDs) (RR), blue LEDs (BB), yellow LEDs (YY) or green LEDs (GG). Cultures grown under fluorescent lamps (FL) were used as control. Selected FL-induced PLBs were cut into 3- to 4-mm sections and incubated on MS medium supplemented with 1.0 mg l⁻¹ BA and 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and grown under RR, BB, YY, GG, or FL. Moreover, FL-differented shoots (15 mm in length with two leaves) were incubated on 1/2 MS medium with 0.5 mg l⁻¹ NAA, and grown under either FL, RR, 10% blue + 90% red LEDs (1BR), 20% blue + 80% red LEDs (2BR), 30% blue + 70% red LEDs (3BR), BB, 80% red + 10% blue + 10% far-red LEDs (RBFr), or 80% red + 10% blue + 10% green LEDs (RBG). Overall, the red light spectrum enhanced induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths, while the blue spectrum promoted differentiation, protein accumulation, and enzyme activities in PLBs, as well as pigment content accumulation in PLBs and developing plantlets. The combination of red and blue LEDs resulted in higher energy efficiency as well as dry weight and enzyme activities in these plantlets.     

Shoot organogenesis in leaf explants of <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla ‘Hyd1’ was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla ‘Hyd1’.     

Micropropagation of <Emphasis Type="Italic">Clitoria ternatea</Emphasis> Linn. (<Emphasis Type="Italic">Fabaceae</Emphasis>)— An important medicinal plant

Summary An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.