De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity

A method is described for producing de novo shoots from leaf derived callus of carnation (Dianthus caryophyllus L.). Plants were regenerated in four steps, viz. callus induction, shoot regeneration, removal of hyperhydricity from regenerated shoots and root development. Callus induction medium contained 2,4-D and BAP. Shoot buds were formed when the callus was further subcultured on 2,4-D- and BAP-containing medium, or MS medium without any growth regulators. The shoots so formed were hyperhydric, bushy in appearance with reduced stem length and watery leaves. The normal conformation of shoots was restored by culturing the hyperhydric shoots onto medium supplemented with GA₃ and bactopeptone. The recovered shoots were rooted on MS medium added with NAA (1 mg/l) or IBA (2 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions after initial acclimation.     

Microsporogenesis and anther culture in carob tree (Ceratonia siliqua L.)

An in vivo study was made on male flowers of carob tree (Ceratonia siliqua L.), in order to establish a correlation between the flower and anther development, and microsporogenesis. In addition studies were conducted to find which phase is more appropriate for anther culture and haploid production. During the development of male flowers, six stages were identified. The male gametophytic cycle begins when flowers are in developmental phase 0, with the formation of the epidermis, endothecium, primary sporogeneous tissue, primary parietal cells and pollen mother cells. During developmental phase I we observed the formation of pollen mother cells, the microspore tetrads, and uni- and binucleate pollen grains. At developmental phase II, uni- and binucleate microspores, and completely formed pollen grains were observed. In developmental phase III we could observe mature pollen grains ready to be released from the anthers as single binucleate pollen grains. Anthers from flowers at developmental phases I and II, with microspores at late uninucleate to early binucleate stage, were cultured in semi-solid Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) combined with one of the citokinins: N⁶-benzyladenine (BA), kinetin (Kin), zeatin (Zea) and thidiazuron (TDZ). To obtain embryogenic calli anthers should be collected from flowers in developmental phase I. High frequencies of callogenesis were obtained, and the best medium for calli induction was MS supplemented with 0.5 mg l⁻¹ 2,4-D + 4 mg l⁻¹ TDZ. The frequency of haploid cells was found to be 17.2%.     

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ Kn, which contained only half concentration of NH₄NO₃. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm²) was transferred to solid media (MS + 0.2 mg l⁻¹ 2,4-D + 5.0 mg l⁻¹ BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.     

Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive (Olea europea L.)

Regeneration via somatic embryogenesis from callus was studied in ‘Nabali’ olive (Olea europea L.). Among different explant sources (leaf blades, leaf petioles, hypocotyls of germinated seeds and roots of germinated seeds), roots gave the highest (46%) callus induction. Somatic embryogenesis was induced from root callus on embryogenesis induction medium (EIM) containing 5.0 μM 2,4-D, 0.5 μM kinetin and 5.0 μM NAA in darkness. Embryo regeneration was studied by transferring the callus from EIM to embryogenesis expression medium (EEM) containing different concentrations (0.0, 5.0, 10.0 and 15.0 μM) of 2-isopentenyladenine (2iP), BA, thiadiazuron (TDZ), zeatin or kinetin. Among the tested concentrations, 2iP at 10.0 μM outperformed the other growth regulators. 2,4-D at 5.0 μM in the EIM was satisfactory for embryogenesis induction. Sucrose at 0.2 M evoked higher embryogenesis than any other concentration of fructose and glucose in EIM, while sorbitol and mannitol at 0.1, 0.2 or 0.3 M reduced embryogenesis significantly and inhibited it totally at 0.4 M. Somatic embryos were rooted by transferring them to hormone-free medium (HFM). About 85% of embryos converted to rooted plantlets, 5% showed secondary embryogenesis and 10% were not developed and died. Rooted plantlets gave 95% survival when acclimatized ex vitro. Acclimatized plantlets developed into whole plants in the greenhouse and they were phenotypically similar.     

Micropropagation of Vitis thunbergii Sieb. et Zucc., a medicinal herb,through high-frequency shoot tip culture

This study describes a protocol for rapid and large-scale in vitro propagation of the valuable medicinal herb Vitis thunbergii Sieb. et Zucc.. Culture conditions influencing shoot proliferation and rooting of the two clones (three- and five-lobed) were examined. Three medium formulations, Murashige-Skoog (MS), Woody Plant Medium (WPM) and Nitsch and Nitsch (NN) medium, were tested for growth of shoot tip culture, and WPM was found to have a superior proliferation rate. The chlorophyll content of leaves was highest in those cultured on NN, followed by those on MS and WPM medium. WPM medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BA) displayed the highest proliferation rate (15–19 nodes or 3–4 shoots per explant). The rooting was optimized using MS medium supplemented with 0.5 mg l⁻¹ napthaleneacetic (NAA) with eight roots, 3 cm long after 1 month of culture. High frequency callus formation was observed in the basal end of explants cultured on NAA-containing medium. Following acclimatization, rooting plantlets were transferred to the plastic house with a 95% survival rate.     

In vitro response of pistachio nodal explants to silver nitrate

The effect of silver nitrate on shoot differentiation and shoot growth was examined in order to improve the regeneration efficiency of pistachio (Pistacia vera L. cv. Kirmizi) in vitro. Nodal explants of in vitro-grown seedlings were used to test various concentrations and combinations of 6-benzyladenine (BA), kinetin (KIN), gibberellic acid (GA₃) and silver nitrate (AgNO₃). Addition of AgNO₃ up to 48.0 μM to the culture medium improved the regeneration frequency and shoot growth, and reduced basal callus formation in all regenerated explants. The highest regeneration frequency (100%) was recorded on Murashige and Skoog (MS) medium containing 9.0 μM BA, 0.2 μM GA₃ and 24.0 or 48.0 μM AgNO₃ in combination. The best proliferation response in terms of both shoot formation and low callus production was obtained in the medium containing a combination of 9.0 μM BA, 0.2 μM GA₃ and 12.0 μM AgNO₃. Regenerated shoots, coming from three cycles of subculturing in proliferation media, were rooted in half-strength MS medium containing 12.0 μM indole-3-butyric acid (IBA). Well rooted plantlets were acclimatized and eventually established in peat and perlite. The development and optimization of an effective micropropagation protocol that is presented in this paper can give an important contribution to improve the quality of pistachio plants and, as a consequence, of orchard production in Middle East countries.     

Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.)

Studies were conducted to improve adventitious bud regeneration in roses (Rosa hybrida L.), specifically to extend the protocol to different genotypes and to initiate production of multiple shoots per explant. The best results were obtained by using a two-stage procedure where excised leaflets were incubated on Murashige and Skoog (MS) (1962) induction medium with 6.8 μM TDZ plus 0.49 μM IBA in the dark for 7 days and subsequently transferred to an MS-based regeneration medium with 2.22 μM BA plus 0.049 μM IBA exposed to a PPFD of 15 μmol m⁻² s⁻¹ PAR. Bud formation capacity was also significantly affected by the genotype and the environment, such as the use of bottom cooling creating a lower RH in the vessel. The addition of silver nitrate to the induction medium also significantly improved the percentage of regeneration in three genotypes tested. Regenerated shoots failed to elongate when transferred to MS proliferation medium containing 0.5 mg l⁻¹ BA, however maximum bud development and elongation were achieved when kinetin in the range 1–2 mg l⁻¹ was used. Elongated shoots were excised and rooted best on zero growth regulator half-strength MS modified medium. Rooted plantlets were acclimatized under greenhouse conditions for evaluation of somaclonal variation.     

Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine

A brief culture of mulberry leaves for 8–10 days on MS medium with 18.17 μM TDZ followed by transfer to 8.88 μM BAP supplemented medium triggered high frequency shoot organogenesis (77.6–89.2%) and favoured shoot elongation in Morus spp. Shoot proliferation was highest in the presence of 2.22 μM BAP with induction of 9.4–10.6 shoots per culture. High frequency of root induction (76.0–86.6%) was observed on medium supplemented with 0.49 μM IBA whereas increase in the level of IBA (4.92 μM) resulted in induction of roots along with development of callus from the base of the shoots. The regenerated plants established in soil at higher frequency in rainy season compared to winter and summer.     

Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing

Multiple shoots were produced from node explants of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) and α-naphthaleneacetic acid (NAA). The highest number of shoots, nine shoots per node, were produced on a medium containing 2 mg l⁻¹ BAP (8.8 μM), 1 mg l⁻¹ kinetin (4.6 μM) and 1 mg l⁻¹ NAA (5.4 μM). Depending on the concentration of BAP and kinetin, NAA either inhibited, stimulated or did not affect shoot multiplication, which also depended on the cytokinin level. Maximum shoot length was obtained from treatments containing 0.5 mg l⁻¹ BAP (2.2 μM) combined with 1 mg l⁻¹ kinetin (4.6 μM) and 0.5 mg l⁻¹ NAA (2.7 μM). The largest leaves of resultant shoots were produced on a medium containing 0.5 mg l⁻¹ each of kinetin (2.3 μM) and NAA (2.7 μM). Transferring in vitro shoots to rooting media containing indole-3-butyric acid (IBA) and NAA produced complete plantlets. The highest rooting percentage was obtained on a medium containing either 1 mg l⁻¹ NAA (5.4 μM) alone or 0.5 mg l⁻¹ NAA (2.7 μM) combined with 2 mg l⁻¹ IBA (9.6 μM), whereas the highest number of roots were produced on a treatment containing both 2 mg l⁻¹ NAA (10.8 μM) and 2 mg l⁻¹ IBA (9.6 μM). Roots elongated most on treatments containing 0.5 mg l⁻¹ of either NAA (2.7 μM) or IBA (2.4 μM). Shoot growth associated with the rooting phase was the highest in response to 2 mg l⁻¹ IBA (9.6 μM) or 0.5 mg l⁻¹ NAA (2.7 μM). Plantlets that survived acclimatization, 82%, exhibited normal growth in soil under greenhouse conditions.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

A liquid 2,4-D pulse increased shoot and root regeneration from leaf explants of adult Prunus rootstocks

Regeneration of adult plant material is one of the main limitations for successful Prunus rootstock transformation. Results herein show that a liquid pulse (90 min) of 2,4-dichlorophenoxyacetic acid (2,4-D) (1.7 μM), applied to leaf explants, greatly improved shoot regeneration in Marianna 2624 (Prunus cerasifera × munsoniana) and Myrobalan 605 AD (P. cerasifera); and induced roots in Adafuel (Prunus × amigdalo-persica) when placed in regeneration medium. Whole leaves and basal leaf explants of Marianna 2624 regenerated shoots in a higher proportion of explants after the pulse (up to 58.9% in whole leaves) than medium or tip leaf segments, whereas the leaf tip was the explant that showed less regeneration. In the regeneration medium, in the presence of BAP, NAA was more effective than 2,4-D. The application of an auxin pulse is a simple method that could enhance adult plant regeneration in commercial rootstocks.     

Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum

An efficient system has been developed for the in vitro plant regeneration of Lilium longiflorum Thunb. by culturing receptacle sections from flower buds. The sections were cultured on one-half MS medium plus 30 g l⁻¹ sucrose, 8 g l⁻¹ agar, 5.4 μM NAA or 4.9 μM IBA plus 2.2 μM BAP. A section size of 3–4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l⁻¹ sucrose. Rooting occurred on one-half MS medium with 1.1 μM NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal flowering plants were produced.     

Factors affecting tissue culture of Damask rose (Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N⁶-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.     

Optimization of somatic embryogenesis in suspension cultures of horsegram [Macrotyloma uniflorum (Lam.) Verdc.]—A hardy grain legume

Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0 μM zeatin and 4.5 μM NAA. Numerous somatic embryoids (26.4%) appeared on MS liquid basal nutrient medium with 5.6 μM NAA and with absence of zeatin during 3 weeks culture. Sustained cell division resulted in the formation of cell aggregates, and then progressed to globular, heart and further if they differentiate properly to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Only a relatively few number of embryos developed into root/shoot when transferred to 0.9 μM GA₃, 15 g/l⁻¹ sucrose and 2.4 g/l⁻¹ gelrite containing medium. Substitution of sucrose associated with the use of l-glutamine gave, in the range of concentrations tested, the strongest enhancement of the embryo growth and development. About 5% of somatic embryos were converted into true-to-type fertile plants.     

Improvement of in vitro micropropagation of Lilium oriental hybrid ‘Casablanca’ by the formation of shoots with abnormally swollen basal plates

Bulb scales of Lilium oriental hybrid ‘Casablanca’ were cultured on MS medium supplemented with cytokinins (kinetin, BA (benzyladenine), TDZ (thidiazuron)). The basal part of bulb scales swelled and formed adventitious shoots with foliage small, leafy bulb scales and abnormally swollen basal plates on the media with cytokinins. Shoots were formed rapidly from the basal parts of bulb scales and became shoot clusters. The medium containing 2.2 μM BA was most favorable in the shoot formation from bulb scales. Cutting shoots into small segments (7–8 mm wide × 12–15 mm length) were cultured on media containing BA and IAA (indole acetic acid) and the segments regenerated many new shoots and formed shoot clusters. The shoot section to shoot cluster cycle method improved adventitious shoot proliferation. The media supplemented with 4.4 μM BA and 5.7 μM IAA. or 8.9 μM BA and 0.6–2.9 μM IAA were effective in allowing the proliferation of shoots from shoot segments under light condition. Sucrose and activated charcoal (AC) improved bulblet growth. Bulblet growth was effectively performed on MS medium containing 60 g/L sucrose. Bulblet growth was also improved by the supplement of AC. The medium with 2.0 g/L AC was most effective for bulblet growth. Normal bulblet growth was stimulated more by the culture of shoots than that of bulb scales. Bulblet weight from shoots reached to an average of over 1100 mg of a bulblet after 3 months in culture on MS medium containing 60 g/L sucrose and 2 g/L AC. Most of the bulblets produced by this method generated stems with several leaves in the green house, after cold treatment at 5 °C for 3 months.     

Embryo induction via anther culture in papaya and sex analysis of the derived plantlets

We investigated the embryo induction of papaya by anther culture, and identified the sex of plantlets derived from embryos using a sex-diagnostic PCR. Anthers, containing approximately 80% uninucleate pollen, were collected from 10 to 14 mm long male flower buds. They were pre-treated on agar (0.8%) or in liquid medium for 1–5 days at 25 or 35 °C, then transferred to agar medium with 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Agar and liquid media used for the pre-treatment contained water only or MS nutrients with or without sucrose (2.0%). On the agar medium, no embryos were induced at any pre-treatment temperature. In the liquid medium at 25 °C, embryos were induced at about 1.0% (rate of anthers forming embryos) in MS medium with sucrose for 3 or 5 days. At 35 °C, embryo induction rate tended to increase up to about 4.0% when anthers were treated in water for 1 day or MS medium with sucrose for 3 or 5 days. The sex of plantlets established through anther culture was analyzed using a sex-diagnostic PCR. All plantlets were determined as female. From these results, we suggest that all plantlets established through anther culture were of microspore origin, and that the anther culture technique is useful for the breeding of female papaya.     

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability were investigated. The growth of shoot tip cultures on agar MS medium containing 2.0 mg l⁻¹ BA was greater than that of similar cultures in liquid MS medium with the same BA concentration. In liquid medium, the combinations of 0.5, 1.0, or 2.0 mg l⁻¹ BA with 0.05 mg l⁻¹ NAA tended to enhance the growth of the cultures. The efficiency of tetraploid induction was assessed by treating shoot tip explants on agar or in liquid MS medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 4, 8, 12, and 14 days. The total number of tetraploids induced on solid medium was 18, but only five in liquid medium. On both media, the colchicine treatment for 8 days gave the maximum level of tetraploid induction. Therefore, it was found that the treatment of shoot tip explants on agar medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 8 days was the most efficient way of inducing tetraploid ginger. Induced tetraploid strains, ‘4× Kintoki’, ‘4× Sanshu’, and ‘4× Philippine cebu 1’, had higher pollen fertility and germinability than the diploid counterparts, i.e., in the diploid strains, pollen fertility ranged from 0.3 to 6.2% and the germination rate from 0.0 to 0.1%, while in the tetraploid strains, pollen fertility ranged from 27.4 to 74.2% and the germination rate from 4.8 to 12.9%.     

Double-phase in vitro culture using sorbitol increases shoot proliferation and reduces hyperhydricity in Japanese pear

This study aimed to improve in vitro shoot proliferation efficiency without inducing hyperhydricity in Japanese pear. The shoot number increased at 2.5–10.0 mg l⁻¹ benzyladenine (BA), while shoot fresh mass increased at 1.0 and 2.5 mg l⁻¹ BA. Different macroelement formulation did not affect shoot proliferation, but adding activated charcoal (AC) to the medium inhibited markedly the production of axillary shoots and biomass and many shoots were hyperhydric. Different carbon sources (CS) significantly increased the shoot number and fresh mass, with the best results for shoot proliferation at 20–30 g l⁻¹ sorbitol. With gelling agents, the shoot number increased at 0.4 and 0.6% agar and 0.3% gellan gum, while fresh mass increased at 0.4% agar. The hyperhydric explants were more than 30% at 0.4–0.6% agar and at any concentration of gellan gum. The improved culture (woody plant (WP) supplemented with 20 g l⁻¹ sorbitol, 0.1 mg l⁻¹ 3-indolyl-butyric acid (IBA), 2.5 mg l⁻¹ BA and 0.8% agar) and double-phase culture (the same medium using a double-phase liquid-gelling agent solidified culture system) produced a higher number of axillary shoots than the conventional culture (1/2MS supplemented with 0.1 mg l⁻¹ IBA, 1.0 mg l⁻¹ BA, 30 g l⁻¹ sucrose and 0.8% agar), moreover, double-phase culture had a higher fresh mass than the other cultures.