Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Recurrent production of plants of black plum, Syzygium cuminii (L.) Skeels, a myrtaceous fruit tree, from in vitro cultured seedling explants

 The epicotyl segments bearing scaly leave(s), excised from in vitro grown seedlings of Syzygium cuminii, produced multiple shoots when cultivated on Murashige and Skoog's (MS, 1962) medium supplemented with different concentrations of BA (0–2 mg l^–1). The optimum response was recorded on the medium containing 1 mg l^–1 BA. An average of 8.6 shoots per explant were produced 60 days after inoculation, following transfer to fresh medium after 30 days. The shoots were excised, and the residual explants were transferred to fresh medium, where they again developed shoots. Up to five such passages resulted in the production of shoots from the repeatedly subcultured original explants. However, during the fifth passage, organogenic response was negligible and the explants turned brown thereafter. Following repeated harvesting of shoots and subculture of the residual explants, an average of 29 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The developed plantlets were planted in soil and transferred to fields after an acclimatization period of 7–8 months. These plants have been thriving well for more than 3 years. The nodal explants excised from in vitro developed shoots and plants also exhibited a similar response when cultured on MS+1 mg l^–1 BA. Thus, a protocol has been developed to raise plants of S. cuminii at any time of the year. Received: 1 December 1998 / Revision received: 1 July 1999 · Accepted: 12 July 1999     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency axillary bud multiplication and ex vitro rooting of Wedelia chinensis (Osbeck) Merr.--a medicinal plant

An efficient protocol was achieved for rapid propagation of Wedelia chinensis (Osbeck) Merr. through axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 8.87 microM) and indole-3-butyric acid (IBA; 2.46 microM) was optimal for axillary bud proliferation, which developed a mean of 8.3 shoots/node. Excision and culture of node segments from in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 days. Excision and culture of nodes in succession enhanced the number of shoots. Shoot multiplication did not exhibit decrease in the number of shoots even at 10th subculture. Nevertheless, the shoots exhibited a tendency towards stunted nature. But reduction of BA to 4.44 or 2.22 microM resumed normal growth of shoots. Half strength MS medium fortified with IBA (2.46 microM) induced the highest number of roots. All in vitro rooted shoots survived in field. Dipping of the basal end of shoots collected from multiplication medium in IBA (2.46 microM) solution for 7 days induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100%). Rooting ex vitro by direct transfer of shoots from multiplication medium exhibited 89.2 per cent survival. Use of commercial sugar and tap water and also the omission of in vitro rooting reduce the propagation cost 50-70 per cent. The protocol enables to harvest more than 50,000 plantlets within 150 days starting from a single node explant.     

In vitro propagation and assessment of genetic stability of micropropagated Samanea saman (rain tree) using microsatellite markers

An efficient in vitro propagation of Samanea saman (rain tree) protocol has been successfully developed using nodal explants from a 20-year-old tree. Higher percentage (76 %) of explants produced up to five shoots per explant on Murashige and Skoog (MS) medium supplemented with 2 mg L^?1 6-benzyladenine (BA), 0.1 mg L^?1 gibberellic acid (GA₃) and 100 mg L^?1 casein hydrolysate after 3 weeks of culture. When explants were subcultured to fresh medium after harvesting first batch of shoots, more shoots could be generated (another eight shoots per explant). Shoot elongation was achieved (3 cm) when shoots were cultured on MS medium supplemented with 0.25 mg L^?1 BA and 0.75 mg L^?1 GA₃. In vitro generated shoots rooted on MS medium fortified with 0.75 mg L^?1 indole-3-butyric acid plus 0.1 % of activated charcoal. A higher percentage of explant response and shoots per explant were obtained on MS medium with BA and GA₃. Each responsive nodal explant yields an average of 15 rooted plants within a period of 10 weeks. Rooted plantlets were successfully acclimatized in green house with a survival rate of 90 %. Micropropagated plants were tested for genetic stability using simple sequence repeats (SSR) markers. Use of the 12 high-resolution SSR markers revealed the exact same genetic profile between the mother tree (donor) and micropropagated plants, suggesting the genetic fidelity of our micropropagation protocol. The same protocol was also used successfully in propagating a “Golden Rain Tree” although response of explant and efficiency of propagation was much lower. This protocol will be useful for germplasm preservation/large scale production of true-to-type clones of desirable genotypes.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of Indian Kino (<Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.) using Thidiazuron

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Micropropagation of Terminalia arjuna Roxb. from cotyledonary nodes

Cotyledonary node explants excised from 21 day old seedlings of T. arjuna produced multiple shoots when cultured on full strength MS or modified MS (1/2 strength major salts and Fe-EDTA) medium supplemented with different concentrations (0.1-1.0 mg/l) of BAP. Maximum 8.9 shoots/explant could be recorded after 30 days of inoculation on modified MS medium supplemented with BAP (0.5 mg/l). A proliferating shoot culture was established by reculturing the original cotyledonary nodes (2-3 times) on shoot multiplication medium after each harvest of the newly formed shoots. Shoots (each having 2-3 nodes/shoot) thus obtained were also used as a source of nodal explant that gave rise to 1-2 shoots when cultured on modified MS+BAP (0.5 mg/l) medium. Thus, 45-55 shoots could be obtained after 60 days of culture initiation from a single cotyledonary node. About 88% shoots rooted well after 15 hr pulse treatment with IBA (1 mg/l) in liquid MS medium followed by transfer to modified MS medium without IBA. About 80% of these plantlets were successfully acclimatized in plastic pots containing sand and soil mixture and 70% plantlets transferred in the field those survived even after 6 months of transplantation.     

Multiple shoot induction and plant regeneration from embryonic axes of cotton

Cytokinins are involved in shoot development of plants. Events of multiple bud formation and shoot development in apical embryonic axes of cotton treated for 2 or 20 days with the cytokinin benzyladenine (BA), were compared with the development of untreated control axes. Meristematic regions (supernumerary vegetative buds) were observed in axes treated for 20 days with BA. An average of 3.4 shoots per embryonary axis was obtained when explants were cultured on medium supplemented with 3 mg l-1 BA. Higher and lower concentrations of the growth regulator yielded fewer shoots per explant. Results shown in this report suggest that BA is directly responsible for re-programming the embryonic apical meristem axes of cotton toward the production of multiple buds and subsequent shoot development. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Feronia limonia</Emphasis> (L.) Swingle from hypocotyl and internodal explants

The hypocotyl and internodal segments from in vitro grown seedlings of Feronia limonia (L.) Swingle (wood apple) were cultivated on Murashige and Skoogs (1962, MS) medium supplemented with N⁶-benzyladenine (BA) or adenine (ADE) or kinetin (KN) at 0.5 to 5 µM. The optimum response was recorded on the medium containing 2 µM BA. An average of 12 and 8 shoots were developed from hypocotyl and internodal explants, respectively, after eight weeks of culture. The shoots were excised, and the residual explants were transferred to fresh medium where again they developed shoots. Up to three such passages resulted in the production of shoots from repeatedly subcultured explants and an average of 24 – 36 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to half strength MS medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). The developed plantlets were successfully transferred to mixture of soil, sand and coco-peat (1:1:1) and hardened in controlled environment. Hardened plants were transplanted to soil in greenhouse.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer through seedling explant cultures

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer was achieved using axenic seedling explant cultures. Isolated nodes (1.0-1.2 cm) and shoot tips (1.0-1.5 cm) cultured in Murashige and Skoog's agar medium containing varying concentrations of TDZ, BA and combinations of 2iP and GA3. Single shoot (0.8-1.2 cm) was regenerated in each culture after 6 weeks. Axillary shoots were then excised and recultured for 8 weeks in medium containing TDZ (0.05 mgL-1) which formed shoots (about 4 in no.; 2 cm) from the basal node. Axillary branches (2) which formed on 60% of these shoots after 10-12 weeks of culture were separated and recultured in the same medium for 8 weeks. Three shoots (0.8-1.0 cm) per culture were regenerated. Shoots of 0.8-1.8 cm length were subcultured on a low cytokinin (0.01 mgL-1 TDZ) regime to induce shoot elongation (2.0-3.5 cm) in 4 weeks. Shoot cuttings were rooted (60%) in the medium containing IBA (1.5 mgL-1). Rooted plantlets established in pots (90%) after hardening resumed normal growth in 3 months.     

In vitro propagation of Dendrobium hybrids using flower stalk node explants

Large-scale in vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly prized commercial cut flower cultivars through shoot multiplication using flower stalk node explants and protocorm-like bodies (PLBs) formation was accomplished. Both hybrids did not exhibit significant differences in initiation, multiplication, rooting, and field establishment. Flower stalk nodes cultured on half strength Murashige and Skoog (MS) medium supplemented with 6.97 microM kinetin (Kn), or 15% coconut water (CW) or 13.3 microM of N6-benzyladenine (BA) evoked bud break. Kn showed better growth of the initiated bud. Excision and culture of the initiated shoots on medium having same amount of Kn developed more than 5 shoots per shoot directly from the base. Subsequent culture enhanced the rate of shoot induction. Transfer of isolated shoots onto 44.4 microM of BA enriched medium displayed induction of more than 6 PLBs from the base within 60 days. PLBs underwent rapid multiplication upon transferral to medium having the same concentration of BA (44.4 microM). Subsequent culture increased the proliferation of PLBs. No decline was observed in the proliferation of shoots as well as PLBs up to 15th subculture. PLBs transferred onto half strength MS medium with 6.97 microM of Kn underwent conversion of more than 90% PLBs to shoots. The shoots were rooted at the best on half strength MS medium with 2 g l(-1) activated charcoal. Survival rate of the plantlets of the two hybrid cultivars after acclimatization was more than 80%.     

Micropropagation of Sesbania sesban from the Mature Tree-Derived Explants

The nodal and internodal explants excised from the orthotropic shoots of Sesbania sesban var. bicolor elicited the development of shoots directly from the explants as well as via an intervening callus phase on Nitsch (N) medium. On benzyladenine (BA) supplemented media, the adventitious shoot buds developed involving a callus phase. An average of 8.9 ± 4.1 shoots developed per nodal explant on N medium containing 0.5 mg dm⁻³ BA in 95 % cultures, whereas 65 % cultures of internodal explants developed shoots with an average of 5.9 ± 3.6 shoots per explant on N medium supplemented with 1.0 mg dm⁻³ BA. On kinetin (Kn) supplemented medium shoots developed directly from the surface of both the explants at all the concentrations tried. Nodal explants on N medium supplemented with 1.5 mg dm⁻³ Kn developed an average of 12.5 ± 7.9 shoots per explant in 100 % cultures, while internodal explants induced an average of 11.6 ± 7.4 shoots per explant in 75 % explants at 0.5 mg dm⁻³ Kn. The in vitro regenerated shoots developed roots when implanted on N medium supplemented with 2 mg dm⁻³ indole-3-butyric acid (IBA), after 30 d of inoculation. The in vitro developed plantlets were initially acclimatized under controlled conditions for four months, prior to their transfer to the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

<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.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

<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.     

一种快速有效的741杨离体叶片再生芽方法

在对杂交品种741杨(Populus alba(P.davidiana×P.simonii)×P.tomentosa)进行农杆菌介导法转基因的试验中发现了一种快速有效的叶片再生芽的方法.首先叶片外植体在培养基Ⅰ(MS培养基添加0.5 mg/L BA和1.0 mg/L 2,4-D)上培养2～3 d,再转移到培养基SH(MSmedium containing 2.0 mg/L of BA and 0.1 mg/L of NAA)上培养10 d,然后再转移到培养基Ⅱ(MSmedium with 0.5 mg/L of BA)上,培养大约5 d之后86.7%的叶片外植体产生的芽,每片叶片外植体(1 cm×1 cm)可产生40～50个芽.但是,如果叶片外植体在培养基Ⅰ上培养的时间长于5 d,再依次转移到培养基SH和Ⅱ上,则叶片会产生大量根.     

Multiple shoot formation from cotyledonary node segments of Eastern redbud

A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 μM BA and 0.5 or 1.0 μM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 μM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.