Micropropagation of globe artichoke (Cynara scolymus L.) from underground dormant buds (“ovoli”)

Summary Side shoots excised from underground dormant buds ofCynara scolymus L. were used as primary explants to establishin vitro cultures. A 3×3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/liter or 2.22, 4.44, 8.88 μM) ofN ⁶-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 μM) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multiplication. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 μM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 μM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Summary Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N⁶-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

A rapid in vitro propagation protocol for Piper barberi Gamble, a critically endangered plant

Summary A protocol is described for rapid multiplication of Piper barberi Gamble (Piperaceae) through shoot tip and nodal explant cultures. Nodal explants with a single axillary meristem showed three times better response with respect to shoot proliferation when compared to shoot tip explants. The best shoot proliferation response of nodal explants was observed with a cytokinin combination of N₆-benzyladenine (4.43 μM) and kinetin (2.32 μM), with 88% bud break. The number of shoot initials (2.4) produced per nodal explant was twice the number of shoot initials (1.2) per shoot tip. An average of 6.9±0.58 adventitious shoots were observed from the proximal end of the internodal explants on Mursashige and Skoog (1962) (Ms) basal medium supplemented with N₆-benzyladenine (2.22 μM) and kinetin (0.46 μM). A multiplication rate of 82 shoots per explant could be achieved after 9 wk of subculturing. The in vitro shoots were rooted on one-half and one-quarter MS basal medium. The shoots rooted on one-quarter MS in the dark produced eight roots with an average root length of 3.36 cm and 98% survival. These plants were transferred to the field with a survival rate of 75%.     

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

Summary The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N⁶-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

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.     

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.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

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.     

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.     

Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Clonal micropropagation of <Emphasis Type="Italic">Crataeva adansonii</Emphasis> (DC.) Prodr.: A multipurpose tree

Summary A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl⁻¹, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl⁻¹, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl⁻¹, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl⁻¹, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.     

An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Tylophora indica (Burm. f.) Merrill, via enhanced axillary bud proliferation from nodal explants collected from young shoots of a two-year-old plant. The physiological effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)], ascorbic acid (AA), different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.6 ± 0.71) of shoots and the maximum average shoot length (5.2 ± 0.31 cm) were recorded on MS medium supplemented with 2.5 μM BA, 0.5 μM NAA and 100 mg/l AA at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with 0.5 μM IBA. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with a 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long-term in vitro conservation of T. indica.     

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

High-frequency induction of multiple shoots and clonal propagation from rhizomatous nodal segments of Houttuynia cordata Thunb.—An ethnomedicinal herb of India

Summary This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 &#x03BC;M) or kinetin (Kn, 18.58 &#x03BC;M) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00&#x00B1;2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 &#x03BC;M) compared to BAP and 6-&#x03B3;-&#x03B3;-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 &#x03BC;M Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 &#x03BC;M Kn or 2.46 &#x03BC;M 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (&gt;20 mm) were successfully rooted on MS medium supplemented with 19.60 &#x03BC;M indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).