High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum

Development of suitable strategy to overcome genotypic limitations of in vitro regeneration in sorghum would help utilize high yielding but poor tissue culture responsive genotypes in genetic manipulation programmes. A factorial experiment was conducted with two explants (immature embryos and inflorescences), eight genotypes (five Sorghum sudanense and three Sorghum bicolor genotypes), three levels of 2,4-D (1 mg l⁻¹, 3 mg l⁻¹, and 5 mg l⁻¹), and two levels of kinetin (0.0 mg l⁻¹ and 0.5 mg l⁻¹). The induced callus was transferred to the regeneration media with factorial combinations of IAA (1.0 mg l⁻¹ and 2.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹ and 1.0 mg l⁻¹). S. sudanense regenerated at significantly higher frequency (38.91%) and produced shoots more intensely (2.2 shoots/callus) than S. bicolor (26.93%, 1.26 shoots/callus). Immature inflorescences regenerated at a much higher frequency (46.48%) and produced significantly more number of shoots (2.71 shoots/callus) than immature embryos (22.35%, 0.99 shoots/callus). Moreover, differences for plant regeneration between genotypes of the same species were minimal when using immature inflorescences. Increase in the 2,4-D concentration in callus induction media exhibited inhibitory effect on callus induction, growth, shoot induction and number of shoots/callus but inclusion of kinetin in callus induction media improved these responses. Use of immature inflorescence explant and inclusion of kinetin in callus induction media could overcome genotypic limitations of plant regeneration to a large extent. The extent of variability, heritability and expected genetic advance was more in plant regeneration traits than in callus induction traits. This indicated that the variability in respect of these attributes in the genotypes may be due to the additive gene action and selection of genotypes for these characters would be rewarding.     

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL^–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA modified Woody Plant Media - BA N⁶-benzyladenine - 2-iP 2-isopentenyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAR photosynthetically active radiation Journal Series No. 8938, Agricultural Research Division, University of Nebraska     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

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     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

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.     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

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.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

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.     

In vitro regeneration of Trifolium glomeratum

In vitro regeneration of Trifolium glomeratum, a leguminous forage species, was attempted through leaf, petiole, cotyledon, hypocotyl, collar and root explants and two media combinations. Root and collar explants showed no callus induction. Medium with 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA) and 0.10 mg dm⁻³ N⁶-benzyladenine (BA) was more effective for hypocotyl explant whereas cotyledon and petiole explant were more responsive to 5.0 mg dm⁻³ NAA and 1.0 mg dm⁻³ BA. Friable, green calli obtained from petiole explant on this medium showed organogenetic potential. Modified root-inducing medium having 0.21 mg dm⁻³ indole-3-acetic acid and 2.5 % sucrose was successful for root induction and plantlets were successfully transferred to field after hardening and Rhizobium inoculation.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.