Lettuce (Lactuca sativa): a species with a high capacity for cadmium (Cd) accumulation and growth stimulation in the presence of low Cd concentrations

Summary

Cadmium (Cd) is a metal pollutant that accumulates in cultivated soils and has detrimental consequences in terms of food safety. Lettuce (Lactuca sativa) can be characterised as having a high capacity to accumulate Cd in its tissues. An analysis of Cd tolerance and Cd accumulation was carried out using two varieties of lettuce (‘Divina’ and ‘Melina’). A wide range of CdCl₂ concentrations was used (0.0, 0.1, 0.6, 3.0, and 15.0 µM CdCl₂). The lowest concentration (0.1 µM CdCl₂) stimulated growth, while the two highest concentrations resulted in a reduction in biomass. Cadmium concentrations were found to be twice as high in roots as in shoots. ‘Divina’ displayed lower concentrations of Cd than ‘Melina’ in nearly all treatments. A strong negative correlation was observed between Cd concentration and Cd tolerance in the roots and shoots (R² > 0.87) of both ‘Melina’ and ‘Divina’. Lettuce grown in the presence of 15.0 µM CdCl₂ had leaf Cd concentrations that were 100-fold higher than the legal maximum level for vegetable products marketed for human consumption, but showed no symptoms of dehydration, chlorosis, or necrosis. This result represents an important alert for lettuce consumers and growers.     

Effect of leaf excision time and age,BA concentration and dark treatments on in vitro shoot regeneration of M.26 apple rootstock

SUMMARY

The effect of benzyladenine (BA) concentrations both during the last proliferating subculture before regeneration (10-222 and, 444 µM) and during organogenesis (11.1 and 22.2 µM), leaf excision time (15 and 30 d from the beginning of the subculture), leafage and dark treatments, on adventitious shoot regeneration of M.26 apple roostock were evaluated. Leaves excised 30 d after the beginning of the last proliferating subculture and grown wkhout BA in thè medium gave the highest percentages of organogenesis, while the number of "regenerated shoots per leaf did not differ significantly among the different BA x leaf excision time combinations. The highest BA concentration (22.2 µM) in the organogeneticmedium produced thehighest percentage of regenerating leaves, with no differences between the lengths and numbers of shoots per regenerating leaf. The first twMjnfurled apical leaves showed a greater regenerative ability than the third and fourth ones, whereas the lengths and numbers of regenerated shoots per leaf were similar. The highest leaf organogenetic ratejyas observed when darkness was imposed at the begirP-ning of the last proliferating subculture and/or at the beginning of the organogenetic phase, but more regenerated shoots per leaf were obtained with darkness provided at the beginning or at the end of the lastproliferating subculture; shoot lengths were similar in all the dark treatments. The great influence onorganogenesis of all the treatments applied in the last proliferating subculture indicates the importance of this stage inpreparing explants for shoot regeneration and thus the possibility of using inductive factors in this phase.     

In vitro regeneration and conservation of wild species of Arachis

Summary

Wild species of Arachis are restricted to South America and generally occur in regions under intensive environmental disturbance. Both in situ and ex situ conservation strategies are required in order to maintain the availability of these genotypes. This work developed in vitro regeneration systems from seed explants of 17 wild species of Arachis from six Sections (Heteranthae, Caulorrhizae, Triseminatae, Erectoides, Procumbentes and Arachis). After seed disinfection, embryonic axes, leaflets and cotyledons were excised aseptically and cultured on Murashige and Skoog (MS) medium supplemented with 8.8 µM, 22 µM or 110 µM 6-benzylaminopurine (BAP). Cultures were maintained in a growth chamber at 28° ± 2°C with a 16 h photoperiod. Regeneration patterns from seed explants were similar among species from all Sections. Embryonic axes produced plants through meristematic amplification or multiple shoot formation, while cotyledons and embryonic leaflets produced shoots at significantly lower frequencies through direct and indirect organogenesis, respectively. Shoots obtained from all explants were transferred to MS medium without growth regulators to induce root formation.     

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus × domestica Borkh.)

Summary

The conventional selection system for apple transformation is based on the selectable marker gene, nptII, encoding antibiotic resistance against kanamycin. We tested an alternative selection system based on the use of D-amino acids using the gene, D-amino acid oxidase 1 (dao1) as the selectable marker, in order to avoid the presence of antibiotic resistance genes in the resulting transgenic apple plants. In addition, dao1 allowed the selection as well as the elimination of dao1-transgenic plants, based on differences in the toxicity of different D-amino acids. Regeneration experiments using apple leaf explants revealed that 2 mM D-serine or D-alanine inhibited shoot regeneration. We performed transformation experiments using the apple cultivars ‘Gala’, ‘Holsteiner Cox’, and a progeny of the apple cultivar ‘Pinova’, and the vector p35S::dao1-intron, containing the dao1 and nptII selectable marker genes. Several shoots regenerated successfully on selection media containing various concentrations of D-serine or D-alanine, but transgenic shoots were not obtained. However, three dao1/nptII transgenic apple lines were obtained after selection with kanamycin, indicating that the vector was functional. Furthermore, we showed that 20 mM D-serine could be used to select dao1-transgenic shoots from non-transgenic in vitro shoots, whereas 13 mM D-isoleucine had the opposite effect.     

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

Micropropagation of bay laurel (Daphne gnidium L.)

Summary

Procedures have been developed for the micropropagation of Daphne gnidium, a shrub species of ecological interest, using explants of juvenile and adult origin. Shoot proliferation rates were significantly affected by both salt formulation and benzylade- nine concentration. Best results were obtained on WP medium with 5 µM BA. The presence of indoleacetic acid in the induction medium improved BA-induced axillary bud proliferation from juvenile explants. Rooting of shoots produced in culture was difficult, especially those of adult origin. Besides an absolute requirement for auxin, calcium concentration and the pH of the medium affected the formation of adventitious roots from regenerated shoots of D. gnidium.     

In vitro induction of chromosome-doubling in cultured shoots of three cultivars of mint (Mentha canadensis L.) treated with colchicine

Summary

To improve the yield and quality of essential oils, chromosome-doubling in mint cultivars was induced by treating shoots with colchicine in vitro. Shoot tips of three mint cultivars (‘68-7’, ‘73-8’, and ‘HU 39’) were cultured in vitro and treated at 2 months using either of two methods to induce chromosome-doubling. Explants were immersed separately in each of three concentrations of colchicine [0.1, 0.2, or 0.3% (w/v)] for 24 h or 48 h. Alternatively, shoots were cultured on solid 1.0× MS (Murashige and Skoog) medium supplemented with one of five concentrations of colchicine (10, 20, 30, 40,, or 50 mg l^–1) for 30 d. After each treatment, ploidy levels were measured by flow cytometry. The results showed that high yields of 4n plants were induced by the immersion of shoots in 0.2% (w/v) colchicine for 24 h (13.3%), or by culturing shoots on MS medium containing 20 mg l^–1 colchicine for 30 d (17.3%). The immersion method, which gave a survival rate of 93.3%, was more convenient and less phytotoxic to produce 4n mint plants. Compared with untreated plants, we observed fewer but larger stomata in chromosome-doubled plants. We also observed significant differences in the size, internode length, leaf length, leaf width, and stem width in chromosome-doubled mint plants.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

Somatic embryogenesis and plantlet regeneration from encapsulated embryos of Selinum tenuifolium

Summary

This paper reports, for the first time, somatic embryogenesis and synthetic seed production in Selinum tenuifolium Wall. Mature leaf explants inoculated in Murishige and Skoog (MS) medium supplemented with 3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), containing 3% (w/v) sucrose and 0.7% (w/v) agar, induced 67% callus. Maximum production of globular structures, their differentiation into embryos and germination, occurred with a combination of 2 µM benzyladenine (BA) and 2 µM indole-3-butyric acid (IBA). To protect somatic embryos and produce synthetic seeds, gel capsules were standardised using a combination of sodium alginate and calcium nitrate concentrations. Gel capsules were most effective when formed with a combination of 3% (w/v) sodium alginate and 100 mM calcium nitrate for 30 min. The addition of MS medium to alginate capsules with 3% (w/v) sodium alginate, 3% (w/v) sucrose, 2 µM BA and 2 µM IBA significantly improved their germination rate to 77.8%, as well as their resulting shoot length (5.6 cm) and root length (7.2 cm), compared to controls (57.8%). Most plantlets (66%) survived under nursery condition. Storage at 4°C for different periods (10 d or 20 d) significantly (P < 0.05) reduced the percentage survival and germination of somatic embryos and artificial seeds compared to controls or 5 d storage.     

In vitro morphogenesis of juvenile Annona cherimola Mill,bud explants

A micropropagation procedure for juvenile cherimoya (Annona clierimola Mill.) is described. Axillary shoot proliferation was obtained after culturing nodal sections with lateral buds in basal medium supplemented with either 0.66 μM BA or 1,36μM zeatin. For root induction shoots were pre-incubated for 7 d in basal medium supplemented with 1 gl^?1 activated charcoal, then cultured for 10 d (7 dark/3 light) on medium with 500 μM IBA, 58.4 mM sucrose, and 200 mg I“1 citric acid. Afterwards, shoots were transferred to the same medium without auxin and with the macroelements at half strength. Using this procedure, 95% of shoots rooted. The survival rate at the end of the acclimatization period was 70%.     

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.     

Influence of macronutrient composition,TIBA and dark treatment on shoot formation and nitrogen content in petiole explants of Senecio × hybridus

Summary

The content of ammonium, nitrate and potassium was varied in the macronutrient solutions intended for formation of adventitious shoots from petiole expiants of Senecio × hybridus. The other components of the macronutrients were according to Murashige and Skoog (1962). The largest number of expiants which formed shoots was obtained when the nitrogen concentration in the Murashige-Skoog solution was lowered from 60 to 30 mM and the potassium concentration from 20 to 15 mM. Addition of 1.0 μM TIBA to the medium as well as the standard addition of 4.44 μM BAP and 28.5 μM IAA favoured shoot formation. Even growth in darkness for two weeks immediately after expiant excision increased shoot number. The nitrogen content in the tissue decreased as the nitrogen concentration in the medium decreased, although an increased concentration in the medium from 60 to 75 mM did not increase the nitrogen content in the tissue. When the potassium concentration was changed from 20 to 15 mM, in a medium with 30 mM nitrogen, the nitrogen concentration in the tissue increased. On the other hand, when using a medium with 60 mM nitrogen, the potassium concentration (30 and 20 mM) did not affect the nitrogen content of the tissue.     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.     

The Effect of Soil pH and Manganese Toxicity Upon the Growth and Mineral Composition of the Hop Plant

Summary

The productivity of ornamental foliage plants is related to their capacity to increase their leaf number and leaf size. In Monstera deliciosa, a change in leaf shape is also a pre-requisite for successful marketing. The aim of this work was to describe the effects of different concentrations of exogenous 6-benzylaminopurine (BAP; 5, 50, 100, or 200 mg l^–1) on the control of both leaf size and leaf shape in M. deliciosa, and the impact of these changes on commercial plant productivity. We found an increase of between 15.4 – 23.1% in the rate of leaf appearance (RLA), which reflected a shortening of the phyllochron, and an increase of between 17.5 – 34.9% in the relative rate of leaf area expansion (RLAE) at most of the BAP concentrations tested. This resulted in higher biomass accumulation in both roots and shoots through an increase of between 5.4 – 7.9% in the relative growth rate (RGR), mainly associated with higher net assimilation rates (NAR; increases from 9.0-fold to 11.0-fold) and increased photoassimilate partitioning to the shoots. The most important result of this work was the early appearance of perforated leaf laminae in M. deliciosa plants sprayed with 50 – 200 mg l^–1 BAP, which made them ready for sale.     

Quality changes in hydroponic lettuce grown under pre-harvest short-duration continuous light of different intensities

Summary

The effect of pre-harvest light intensity on the quality of hydroponically-grown lettuce (Lactuca sativa var. capitata L.) was studied by growing lettuce under 48 h of continuous illumination delivered by red- or blue-light-emitting diodes (LEDs) with a red:blue ratio of 4.0. Four light intensity treatments (50, 100, 150, or 200 µmol m^–2 s^–1) were applied. The results showed that the nitrate concentrations in lettuce shoots decreased significantly after treatment with 48 h of continuous light, while the contents of soluble sugars and vitamin C increased substantially. It was observed that the effect of pre-harvest short-duration continuous light (PSCL) on improving lettuce quality was significantly influenced by the intensity of the light. The decrease in nitrate concentration and the increases in soluble sugars and vitamin C contents were relatively low at a light intensity of 50 µmol m^–2 s^–1, but increased gradually as the light intensity increased from 50 µmol m^–2 s^–1 to 200 µmol m^–2 s^–1. However, the marginal benefit of increased light intensity in lowering nitrate concentration and increaseing vitamin C content declined rapidly when the light intensity increased beyond 100 µmol m^–2 s^–1. In conclusion, PSCL offers an effective method by which to improve the quality of lettuce, and a light intensity of between 100 µmol m^–2 s^–1 – 150 µmol m^–2 s^–1 is the economic optimum.     

Fruiting position,mineral concentration and incidence of physiological pitting in ‘Hayward’ kiwifruit

Summary

‘Hayward’ kiwifruit (Actinidia deliciosa (A. Chev) C. F. Liang et A. R. Ferguson) were sampled to identify populations of fruit with differing fruit mineral concentrations and levels of the storage-related disorder physiological pitting. Fruit were taken from different shoot types at different locations within vines. Fruit from short shoots near the tips of canes, in an area of the vine with low leaf:fruit ratios, had low fruit calcium concentrations and more pitting. In contrast, fruit from long shoots and with high leaf:fruit ratios near the base of canes, had high concentrations of calcium and less pitting. At the more distal positions along canes in an area of the vine with low leaf:fruit ratios, increasing leaf:fruit ratios on individual fruiting shoots led to higher inflow of calcium to the fruit. No such relationship was found when fruit were sampled from shoots near the base of canes, an area of the vine with relatively high leaf:fruit ratios. Inconsistent relationships were found between fruit soluble solids concentrations and calcium, probably due to different mobility of calcium and carbohydrates within the vines. Fruiting position and the associated leaf area are a source of variability in mineral concentrations of fruit, and by consequence, in the incidence of physiological pitting in ‘Hayward’ kiwifruit, and should be considered when developing sampling techniques.     

Orange varieties as interstocks increase the salt tolerance of lemon trees

Summary

We investigated the ability of interstocks to increase salt tolerance in lemon trees. We compared 2-year-old ‘Verna’ lemon trees [Citrus limon (L.) Burm.; VL] grafted on Sour Orange (C. aurantium L.; SO) rootstock either without an interstock (VL/SO), or interstocked with ‘Valencia’ orange (C. sinensis Osbeck; VL/V/SO), or with ‘Castellano’ orange (C. sinensis Osbeck; VL/C/SO). Trees were grown under greenhouse conditions and supplied with nutrient solutions containing 0, 30, or 60 mM NaCl. Reductions in leaf growth caused by salt treatment were greatest in non-interstocked (VL/SO) trees, followed by VL/C/SO trees, and were the least in VL/V/SO trees. Although the levels of Cl^? and Na⁺ ions in the roots and stems were not affected by either interstock, leaf concentrations of Cl^? and Na⁺ were higher in VL/SO trees than in VL/C/SO or VL/V/SO trees, suggesting that an interstock in Citrus trees could limit the uptake and transport of such ions to the shoots. Saline-treated VL/SO trees also tended to have the lowest shoot:root (S:R) ratios; so, overall, there was a negative relationship between S:R ratio and leaf Cl- ion concentration. Leaf transpiration (E_leaf) may also be involved in the reduction in leaf Cl^? concentration, as interstocked trees had lower E_leaf values at mid-day than non-interstocked trees. Salinity increased leaf concentrations of Ca²⁺ in VL/C/SO trees and increased both leaf K⁺ and N concentrations in all trees, regardless of interstock. Salinity reduced leaf water potentials and osmotic potentials, such that leaf turgor was increased in all trees.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

In vitro propagation of GF677 hybrid rootstock (Prunus persica × Prunus amygdalus) from mature cotyledons

Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l^?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.