Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants

Transgenic muskmelon (Cucumis melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterium tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanaymcin resistance. After co-cultivation for three days, expiants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3–5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confirmed that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - IAA indole 3 acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NPT II neomycin phosphotransferase II     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation of <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l⁻¹ hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Plant regeneration from stem cortex expiant and protoplast cultures of Brassica juncea (mustard)

Summary Plant regeneration from stem cortex explants of 13 genotypes of Brassica juncea was assessed. Regeneration was strongly affected by genotype, as up to 50.6 shoots were produced per 100 calli of the most responsive line (Blaze), whereas no shoots were obtained from less responsive lines (Zeml, Vniimk351). Blaze was chosen for B. juncea stem cortex protoplast isolation. After one week of culture, 11–14% of the cells had divided, and about 0.002% produced 1–2 mm colonies within 6 weeks. Up to 7% of these colonies gave rise to shoots upon transfer to plant regeneration medium.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BAP 6-benzylaminopurine - FDA fluorescein diacetate - IAA indole acetic acid - IBA indole butyric acid - 2ip 2-isopentenyl-adenine - NAA 1-naphthaleneacetic acid - TD thidiazuron     

A rapid and efficient method for in vitro shoot organogenesis and production of transgenic Bacopa monnieri L. mediated by Agrobacterium tumefaciens

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Bacopa monnieri L. (Scrophulariaceae), a plant well known for its medicinal properties. Leaf explants were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), and in combination with either indole-3-acetic acid (IAA) or napthalene-3-acetic acid. A combination of BAP (17.80 μM) and IAA (2.28 μM) maximized shoot initiation (85.2 ± 2.43) with greatest shoot length (2.8 ± 0.22), and was obtained directly from leaf explants without an intervening callus phase. Leaf segments from in vitro grown plants were co-cultivated with Agrobacterium tumefaciens LBA4404 harboring pCAMBIA1301 with ?-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes. The co-cultivated explants were transferred to selective shoot induction and elongation medium. The elongated hygromycin-resistant shoots were subsequently rooted on MS medium supplemented with 4.9 μM indole-3-butyric acid and 25 mg/l hygromycin (SSRM). Successful transformation was confirmed by monitoring histochemical GUS activity during shoot elongation and PCR analyses using uidA- and hpt-specific primers. Integration of hpt into the genome of transgenic plants was also verified by Southern blot analysis. The highest transformation efficiency achieved was 70.6%, with an average of 10.4 ± 0.15 transgenic plantlets per explant using the present transformation system. Therefore, these highly efficient and rapid regeneration and transformation systems create significant potential for engineering of B. monnieri with a view to detailed biomolecular analyses or for further enhancement of its medicinal properties.     

Somatic embryogenesis from leaf explants of Australian fan flower,Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2–0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium—from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and -naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.Abbreviations ABA Abscisic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -Naphthaleneacetic acid - PIPES Piperazine-N, N-(2-ethanesulfonic acid) Communicated by R.J. Rose     

Plant regeneration from cotyledonary node explants of mungbean (Vigna radiata (L.) Wilczek)

Sexually-mature mungbean (Vigna radiata (L.) Wilczek) plants were efficiently regenerated from cotyledonary node explants. The explants were capable of directly developing multiple shoots on basal media devoid of any growth regulators. The shoot multiplication was influenced by media composition, growth regulators, age of donor seedling and explant type. The explants with both the cotyledons attached to the embryonic axis excised from 4-d-old seedlings, produced the highest number of shoots (5 or 6) in 100% of the cultures within 2 weeks on B₅ basal medium (BBM) containing BAP or 2-iP, respectively, (at 5x10^–7M) and 3% sucrose. Shoots elongated and developed better using BAP. Increasing micronutrients, carbohydrate and nitrogen levels in the medium above the original formulation of B₅ basal medium appeared to be of no benefit for increasing the number of shoots. The shoots were rooted on basal MS medium or MS containing 10^–6 of NAA, IAA or IBA. This protocol was found applicable to six other cultivars of mungbean. One hundred rooted shoots were successfully established in soil in the glasshouse, where 90% of them survived. The regenerated plants flowered precociously, but produced normal pods and viable seeds.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - 2-iP 6- — -dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium - B₅ Gamborg et al. (1968) medium - C medium MS salts + B₅ vitamins     

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which -glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l^–1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional -glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l^–1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.     

In vitro regeneration of Ruscus hypophyllum L. plants

Callus cultures were established from stem explants of Ruscus hypophyllum on a modified basal medium of Murashige and Skoog (1962) supplemented with 1 mg l^-1 2,4-D+0.1 mg l^-1 BAP. The optimal 2,4-D concentration for promoting shoot bud formation and growth was 0.05 mg l^-1 along with 0.5 mg l^-1 BAP. Sixty percent of rootless shoots produced flowers on the regenerating medium. Rooting was induced when shoots were transferred to half strength MS inorganic salts supplemented with 2 mg l^-1 IBA. Eighty percent of plants transferred to soil have survived.     

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL^–1 picloram + 0.01 mgL^–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL^–1 BAP plus either 0.2 mgL^–1 picloram or 0.1 mgL^–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - TDZ thidiazuron     

In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd.

In vitro propagation was initiated from 2-week-old and 7-month-old explants of Acacia mangium. Juvenile explants (2 week-old) of 5- to 10-mm lengths composed of two leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 or 2.0 mg L^-1 6-benzyladenine (BAP). After 6 weeks, most explants had formed a large cluster of 14–18 axillary shoots produced by prolific branching of the primary axillary shoot after elongation. The maximum multiplication rate (40) was obtained in the first subculture; the rate decreased to 10–20 in the second one. The mean length of shoots was not significantly affected by BAP concentrations during the subsequent cultures. Rooting ability of juvenile explants was greatly affected by BAP concentrations used in the multiplication medium. When both types of explants were multiplied on a MS medium containing 1.0 mg L^-1 BAP and transferred to a half-strength MS medium containing 0.05 mg L^-1 IBA, only 10% of the juvenile explants were rooted versus 70% of the 7-month-old explants. Rooted plants transferred onto artificial substrate were all nodulated, when inoculated with a specific Bradyrhizobium sp. strain.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains

Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM Agrobacterium infection medium - CIM callus-inducing medium - CTAB cetyltrimethylammonium bromide - 2,4-D 2,4-dichlorophenoxy-acetic acid - GUS ß-glucuronidase - hph hygromycin phosphotransferase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip N -(2-isopentenyl) adenine - NPTII neomycin phosphotransferase II - RIM root-inducing medium - 35S cauliflower mosaic virus 35S promoter - SIM shoot-inducing medium     

In vitro multiplication and field establishment of Adhatoda beddomei C. B. Clarke,a rare medicinal plant

Clonal propagation of Adhatoda beddomei C.B. Clarke (Acanthaceae), a rare medicinal shrub, was achieved through callus-free axillary meristem proliferation from stem node explants of field-grown plants cultured in SH medium. Shoot multiplication was a function of cytokinin activity but sustained growth of the shoots was dependent on the synergistic effect with the auxin, IAA. An optimum number of 5–10 shoots per explant were obtained in 6 weeks using 3.0 mg.l^–1 BAP, 0.5 mg.l^–1 2-ip and 1.0 mg.l^–1 IAA, Upon subculture, vertical halves of the precultured node with the differentiated shoots yielded a larger aggregate number of shoots (23–27) than the uncut precultured node left intact (15–17). Shoot multiplication was rapid and consistent over prolonged periods when the hormonal concentrations were reduced to 1.0 mg.l^–1 BAP and 0.2 mg.l^–1 IAA during subculture, and reculture of the nodal explants derived from shoot cultures. Rooting of 3–5 cm shoots thus obtained was greatly accelerated in stationary liquid medium containing 0.2 mg.l^–1 IBA or IAA. Hardening of the rooted plantlets in the humidity chamber was essential for high frequency (95%) survival. Micropropagated plants established in the field flowered after fifteen months and were free from apparent defects in cytological, growth and flowering characteristics.Abbreviations SH Schenk and Hildebrandt (1972) basal medium - BAP 6-benzylaminopurine - 2-ip 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid