Anatomy and morphology of photinia (Photinia × fraseri Dress) in vitro plants inoculated with rhizobacteria

Photinia × fraseri Dress (photinia) is a woody plant with high ornamental value. The anatomy and morphology of micropropagated photinia inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense and Azotobacter chroococcum, in combination with pulses of 49.2 μM indole-3-butyric acid during rhizogenesis, were characterized using light and electron microscopy. Leaves of inoculated in vitro plants showed better development than those subjected to auxin control only. All inoculated treatments, independent of the bacterial strain used, had leaves with two layers of palisade parenchyma, a thick cuticle and linear unicellular trichomes. There was no proliferation of undifferentiated tissue in any treatment and the plants showed shoot–root vascular connections. Ex vitro leaves and in vitro plants inoculated with Azospirillum brasilense Cd and Azotobacter chroococcum 42 had large stomata with elliptic aperture radially surrounded by small stomata on the abaxial foliar surface. In addition, plants of these treatments had a large root hair zone over the root surface. Bacteria were only observed on surfaces of root hairs. The results suggest that the structural changes induced by bacterial inoculation of photinia in vitro plants could lead to better adaptation to ex vitro conditions after transplanting.     

Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.)

Plant root development can be largely affected through the association of roots with plant growth-promoting rhizobacteria (PGPR). However, little is known about the identity of plant genes enabling such PGPR-plant root associations. Differences in the responsiveness to PGPR among cultivars suggest genetic variation for this trait within germplasm. In this study, two genotypes of common bean (Phaseolus vulgaris L.), BAT477 and DOR364, were identified showing contrasting responsiveness in root development to inoculation with the PGPR Azospirillum brasilense Sp245. Inoculation with an A. brasilense Sp245 mutant strain strongly reduced in auxin biosynthesis or addition of increasing concentrations of exogenous auxin to the plant growth medium, indicated that the differential response to A. brasilense Sp245 among the bean genotypes is related to a differential response to the bacterial produced auxin. To further assess the role of the plant host in root responsiveness, a population of Recombinant Inbred Lines (RILs) of the DOR364×BAT477 cross was used to evaluate the efficacy of exogenous auxin on root development. We detected significant phenotypic variation among the RILs for basal root formation during germination upon addition of auxin to the growth medium. Genetic analysis revealed two quantitative trait loci (QTLs) associated with basal root responsiveness to auxin of which one explained 36% of the phenotypic variation among the RILs. This latter QTL mapped to the same location as a QTL for root tip formation at low P, suggesting that the host effect on root responsiveness to IAA interacts with specific root development. Also, significant correlations between basal root responsiveness to auxin and growth, root tips and root dry weight at low P were identified. To our knowledge, this is the first report on QTL detection for root responsiveness to auxin.     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.     

A three-stage micropropagation system for a novel color morph of the wildflower, <Emphasis Type="Italic">Porteranthus trifoliatus</Emphasis> (L.) Britt

A three-stage micropropagation system was devised for Porteranthus trifoliatus ‘Pink Profusion’, a cultivar distinguished from the wild type by its pink flowers, purple stems and darker reddish leaves. Nodes of young shoots were used as explants, disinfested, and placed on several different Murashige and Skoog’s (MS) media formulations containing two levels of indole-3-butyric acid (IBA) and four levels of benzylaminopurine (BAP) in a factorial combination. An optimal number of commercially-usable shoots was achieved with BAP concentrations between 10 μM and 30 μM, while the addition of IBA made no significant difference in the number of shoots produced. Proliferated shoots could be rooted ex vitro without auxin treatment.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

Effects of short light-dark regimes on in vitro shoot rooting of some fruit tree rootstocks

Experiments were carried out to evaluate the effects of 4/2 light-dark cycles (4 h of light followed by 2 h of dark) on the rooting responses of shoots cultivated in vitro of the fruit tree rootstocks GF 677 (peach × almond hybrid), Mr.S. 2/5 (Prunus cerasifera), MM 106 (apple Nothern Spy × Paradise M1) and BA 29 (Cydonia oblonga). Under this light regime rooting percentage of GF 677, Mr.S. 2/5 and MM 106 shoots reached 100 % as in the control treatment (16/8), while in BA 29 shoots, short light-dark cycles increased rooting response by about 65 %. Moreover, the shoots of all rootstocks submitted to the 4/2 cycle showed an appreciable increase in root number and length, and an earlier root emergence of about 4 – 5 d compared to the 16/8 cycle. Finally, rooting percentage of BA 29 shoots submitted to the 4/2 light regime and treated with 0.2 mg dm⁻³ indolebutyric acid (IBA), was equal to that reported with 0.4 mg dm⁻³ IBA under the 16/8 regime, indicating that the former light regime also amplified the rhizogenic effect of auxin.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro Propagation of Cloudberry (Rubus chamaemorus)

The purpose of this study was to establish conditions for micropropagation of cloudberry (Rubus chamaemorus L.). Cultures were initiated from meristem cultures. When cultures were subcultured from clusters of 3–5 shoots, approximately 70 and 50 shoots were produced per cluster within 6 weeks at 8.9 μM BAP for the female cv. Fjellgull and the male cv. Apollen, respectively. Addition of 5.5 μM GA₃ reduced the number of shoots. Auxins (IBA, NAA) promoted root development in vitro, but inhibited formation of new shoots. However, as much as 85% of shoots rooted without auxin treatment when planted in a peat:sand (80:20 v/v) mixture. Some of the male plants regenerated from shoot tip cultures flowered in the greenhouse within a year after transfer to soil.     

Thidiazuron induced high frequency axillary shoot multiplication in Psoralea corylifolia

The effect of thidiazuron (TDZ) was studied on in vitro axillary shoot proliferation from nodal explant of Psoralea corylifolia - an endangered medicinal plant. Proliferation of shoots was achieved on Murashige and Skoog (MS) medium supplemented with 0.5, 1, 2, 3, 4 and 5 μM TDZ. The maximum number (13.6 ± 1.4) of shoots per explant were obtained from nodal segment cultured on 2 μM TDZ for 4 weeks and this increased to 29.7 ± 2.1 on hormone free MS medium after 8 weeks. The in vitro proliferated and elongated shoots were transferred individually on a root induction medium containing 0.5 μM indole-3-butyric acid (IBA) and within 4 weeks 4.5 ± 0.5 roots per shoot were produced. The regenerated plantlets were transferred to 1:1 soil and vermiculite mixture and acclimatized with 80 % survival rate. Fully acclimatized plants were grown in garden soil in greenhouse and their morphological and physiological parameters were comparable with seedlings.     

Micropropagation of a <Emphasis Type="Italic">Casuarina</Emphasis> hybrid (<Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> L. × <Emphasis Type="Italic">Casuarina glauca</Emphasis> Sieber ex Spreng) following facilitated seed germination

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot regeneration of the medicinal plant meadowsweet (<Emphasis Type="Italic">Filipendula ulmaria</Emphasis> (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA₃), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA₃. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde

Caulogenic responses of various explant types from 12-month-old plants of Hemidesmus indicus were tested. Second and third visible nodes (0.5 cm) from the apex and root segments (0.5 cm) were the most and least regenerative respectively, with the formation of 9.37 and 2.6 shoots in 4 weeks on half strength MS medium supplemented with 2.22 μM BA and 1.07 μM NAA and 4.44 μM BA and 2.69 μM NAA respectively. Caulogenic ability of the nodes decreased with increasing maturity. Shoot buds initiated upon the young nodes on day 10 developed into 7.2 cm long shoots within 4 weeks thereby making a shoot elongation phase unnecessary. Nodal explants of the in vitro raised shoots subcultured in the same medium produced 9.32 shoots of 7.1 cm length in 3–4 weeks, similar to those of the mature plant-derived nodes. Multiplication through subculture of the nodes up to 25 passages of 4 weeks each was achieved without decline. Shoot cultures were rooted in quarter salt strength MS medium containing 9.8 μM IBA and the rooted plants were hardened for establishment in pots at 96% rate. Four months after establishment, the micropropagated plants were stable and showed uniform morphological and growth characteristic. After 12 months of cultivation in the field, on an average micropropagated plant consisted of 4–5 shoots, 5–8 branches per shoot and increased root biomass (13.5 g) compared to the poor growth (single shoot and 2–3 branches) and root production (4.6 g) values obtained with plants raised from conventional rooted stem cuttings. The concentration of the root specific compound, 2-hydroxy 4-methoxy benzaldehyde per plant was 2–3 fold higher in micropropagated plants though on unit dry root biomass (0.12% per g dry wt) basis it remained the same between two sources of plants. This revised version was published online in June 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.     

<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.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.