In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

<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.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Indirect shoot organogenesis from leaves of Dieffenbachia cv. Camouflage

A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N ⁶-(Δ²-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and morphology have been observed in regenerated plants.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

High-frequency plant regeneration via adventitious shoot formation from deembryonated cotyledon explants of Sesamum indicum L.

Sesamum indicum L. was used as an important oil crop in the world. An efficient protocol for in vitro plant regeneration via adventitious shoot formation from deembryonated cotyledon explants isolated from mature seeds of sesame is developed. Optimal medium for direct adventitious shoot formation was Murashige and Skoog (MS) medium with 22.2 μM 6-benzylaminopurin (BA) and 5.7 μM indole-3-acetic acid (IAA). Abscisic acid (3.8 μM ABA) and AgNO₃ (29.4 μM) were effective in enhancing the frequency of adventitious shoot formation. Preculture of cotyledon explants on high sucrose concentration (6–9%) for 2 wk and subsequent transfer to 3% sucrose enhanced the frequency of adventitious shoot induction. Root formation from the adventitious shoots was easily achieved on MS medium containing 2.7 μM of α-naphthalene acetic acid (NAA). Regenerated plantlets were acclimatized on sand and peat moss (1:1), showing 95% survival with subsequent flowering and seed set. We established the high-frequency plant regeneration via adventitious shoot formation in S. indicum L.     

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.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

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.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Micropropagation of curry leaf tree

Murraya koenigii (curry leaf tree) is cultivated for its aromatic leaves which are used as condiment. Nodal cuttings from mature curry leaf plants cultured in Woody plant basal medium (WPM) supplemented with 4.4 μM benzyladenine (BA) and 4.65 μM kinetin produced 12–30 multiple shoots per node by the eighth week of inoculation. The shoots easily rooted in vitro in woody plant medium contained naphthalene acetic acid 1.35 μM NAA. Ninety percent of the plants survived transfer to a hardening chamber and were transferred to the field after three months. In vitro-developed shoots were also rooted ex vitro by dipping in 2.46 μM indole-3-butyric acid for one minute. They were transplanted to sand in a hardening chamber with 70–80% relative humidity and a temperature of 28±2 °C. Eighty to ninety percent of the ex vitro-rooted plants survived and were transferred to the field after 3 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenesis,embryogenesis, and synthetic seed production in <Emphasis Type="Italic">Arnebia euchroma</Emphasis>—A critically endangered medicinal plant of the Himalaya

Summary This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas leaf-derived callus showed simutaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per culture) in 1 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 μM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1∶1∶1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies, and in long-term storage of elite genotypes through synthetic seed production.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Efficient Somatic Embryogenesis in Alstroemeria

In Alstroemeria high frequencies of compact embryogenic callus (CEC) induction (40%) and friable embryogenic callus (FEC) induction (15%) were obtained from nodes with axil tissue cultured first on a Murashige and Skoog (MS) medium supplemented with 10 μM thidiazuron and 0.5 μM indole-3-butyric acid and after that on a Schenk and Hildebrandt (SH) medium supplemented with 9.1 μM 2,4-dichlorophenoxy acetic acid and 2.2 μM benzylaminopurine (BA). Both types of callus were maintained on modified MS medium supplemented with 20.8 μM picloram. CEC and FEC formed somatic embryos and subsequently plants when transferred to MS medium supplemented with 2.2 μM BA. Plants were produced after 12 weeks (CEC) or after 16 weeks (FEC) of culture. Regenerated plants were established in the greenhouse and flowered normally.     

Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.