Protease excretion during pineapple micropropagation in temporary immersion bioreactors

Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.     

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

Summary Success has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl⁻¹) + ascorbic acid (50 mgl⁻¹) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.     

In vitro propagation of limonium wrightii (Hance) Ktze. (Plumbaginaceae), an ethnomedicinal plant, from shoot-tip, leaf- and inflorescence-node explants

Summary Rapid in vitro propagation of Limonium wrightii (Hance) Ktze. (Plumbaginaceae), an endangered medicinal plant, was achieved by culturing the shoot-tip (primary and lateral), leaf- and influorescence-node explants. MS (Murashige and Skoog, 1962) medium supplemented with 8.87 μMN⁶-benyladenine (BA) and 1.07 μM α-naphthaleneacetic acid (NAA) supported induction of adventitious shoots from the shoot-tip, inflorescence-node and middle and basal parts of leaf explants after 60 d of culture. Adventitious shoots were multiplied by subculturing on MS medium supplemented with BA (2,21–17.75 μM) in combination with NAA (1.07 μM). The percentage of explants forming shoots and the average number of adventitious shoot buds produced per explant were stimulated by increasing the strength (1/4x, 1/2x, 1x, 2x) of the MS medium. Shoots were rooted on MS basal medium with 4.92 μM indole-3-butyric acid. Plantlets with a morphologically normal appearance produced from adventitious shoots were transferred to soil and acclimated in the growth chamber for 1 mo.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

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.     

Effect of growth regulator and culture conditions on shoot multiplication and rhizome formation in ginger (Zingiber officinale Rosc.) in vitro

Summary Shoot multiplication of Zingiber officinale cv. V₃S₁₈ was achieved by meristem culture on a Murashige and Skoog (MS) basal medium supplemented with 26.6 μM 6-benzylaminopurine (BA), 8.57 μM indole-3-acetic acid (IAA), and 1111.1 μM adenine sulfate and 3% (w/v) sucrose. In vitro rhizome formation from in vitro-raised shoots was achieved on MS medium supplemented with 4.44 μM BA, 5.71 μM IAA, and 3–8% (w/v) sucrose after 8 wk of culture. Cultural variations such as photoperiod, carbohydrate, nutrient composition, and growth regulators were tested for the maximum yield of rhizomes. Among the different photoperiods used, a 24-h photoperiod helped in the formation of more rhizomes as compared with other photoperiods. Of the different carbohydrates used, sucrose helped to achieve rhizome formation as compared to other carbohydrates. The microrhizomes sprouted in a soil mixture within 2 wk of planting. The sprouted plantlets survived under field conditions with normal growth.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots

Cultured shoots ofRosa ‘Improved Blaze’ were used to determine the effects of sucrose and inorganic nitrogen on adventitious root formation. Shoots grown in media containing high sucrose concentrations (146.07–262.93 mM) produced more and longer roots than those grown in media containing 0–87.64 mM sucrose. This response to sucrose was related to the metabolism of sucrose rather than its osmotic properties since the use of mannitol and 3-O-methyl-α-D-glucopyranoside as osmotic substitutes did not reproduce the effect on rooting. The number and length of roots increased when the shoots were grown in media with the nitrogen concentration of the Murashige-Skoog (MS) salt formulation reduced from 60 to 7.5 mM. Neither nitrate (NO ₃ ⁻ ) nor ammonium (NH ₄ ⁺ ) alone at any of the concentrations tested had the effect on rooting that both had together in the ratio of the MS salt formulation. When the sucrose and nitrogen concentrations were both varied, the greatest rate of root initiation occurred on shoots grown in media with a high sucrose to nitrogen concentration ratio.     

Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae)

Summary Embryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM α-naphthaleneacetic acid (NAA), 4.4 μM 6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4μM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.     

An efficient micropropagation system for <Emphasis Type="Italic">Eclipta,alba</Emphasis>—A valuable medicinal herb

Summary An efficient rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Eclipta alba (Asteraceae) by enhanced axillary shoot proliferation in cotyledonary node segments was designed. The medium type, various carbon sources, plant growth regulators, and coconut water markedly influenced in vitro propagation of Eclipta alba. An in vitro plantlet production system has been investigated on Murashige and Skoog (MS) medium with the synergistic combination of benzyladenine (4.4μM), kinetin (4.6μM), 2-isopentenyladenine (4.9μM), gibberellic acid (1.4μM), 5% coconut water, and 3% sucrose which promoted the maximum number of shoots as well as beneficial shoot length: Subculturing of cotyledonary node segments on a similar medium enabled continuous production of healthy shoots with similar frequency. Rooting was highest (94.3%) on full strength. MS medium containing 9.8 μM indolebutyric acid. Micropropagated plants established in garden soil, farmyard soil, and sand (2∶1∶1) were uniform and identical to the donor plant with respect to growth characteristics as well as floral features. These plants grew normally without showing any morphological variation.     

Photoperiodically independent flowering of Pharbitis nil plants regenerated from flower buds

Summary Flower buds and anthers of the short-day plant Pharbitis nil were treated either with thermic shock (7 or 35°C) or osmotic/trophic shock (12% sucrose) for 24 h. Explants were transferred either to Murashige and Skoog medium (MS) with addition of 6-benzylaminopurine (BA; 4.4μM) and 6% sucrose or to the same growth medium containing 22 μM BA and 3% sucrose. Both media were supplemented with α-naphthaleneacetic acid (NAA; 0.55 μM). Osmotic/trophic shock stimulated the occurrence of shoots on flower buds grown on medium containing 22 μM BA. Thermic shock (7 and 35°C) inhibited this process on both types of explants. Regenerated plantlets were transferred to MS medium supplemented with 6% sucrose, gibberellic acid (GA₃; 1.44μM), NAA (0.55 μM) and Ca²⁺ (0.66 mgl⁻¹). After 3–4 wk they were able to produce flowers without photoperiodic induction.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> roxb

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microtuttings on a peat moss, soil, and compost mixture (1∶1∶1). About 52% of plantlets were successfully acclimatized and established in pots.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

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.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.