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.     

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.     

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.     

Direct shoot organogenesis on shoot apex from seedling explants of Capsicum annuum L.

In vitro direct multiple shoot formation from seedling explants of Indian high pungent varieties of Capsicum annuum cv. Arka Abhir (AA) and Arka Lohit (AL) was successfully obtained. We were able to induce regeneration potency in these varieties by inverting the explant. Aseptically grown seedling explants with decapitated roots, apical meristem and cotyledonary leaves were inoculated in an inverted position in bud induction medium comprising of Murashige and Skoog's basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid (MES) buffer along with 26.63 μM benzyl adenine (BA), 2.28 μM indole-3-acetic acid (IAA) and 10 μM silver nitrate. Profuse shoot bud induction with 20–25 shoot buds per explant was obtained. Supplementation of phloroglucinol in the bud induction medium resulted in 17 and 18% enhancement in bud induction response in Arka Abhir and Arka Lohit variety, respectively on the inverted hypocotyls. Auxin transport inhibitor tri-iodo benzoic acid (TIBA) in the bud induction medium resulted in induction of buds in a shorter period of 40–45 days when compared to bud induction (BI) medium which takes 55–65 days for bud induction. These buds were transferred to MS medium containing 2.8 μM gibberellic acid and 10 μM silver nitrate resulting in elongation of shoot buds. Transfer of shoots to MS basal medium induced rooting to produce plantlets. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. annuum tissues.     

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.     

An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings

The possible application of some less commonly used in vitro growth regulating compounds is outlined. A number of treatments were applied to determine the best way of inducing in vitro shoot proliferation and rooting on a modified Driver–Kuniyuki [HortScience 19 (1984) 507] basal medium of lemon (Citrus limon (L.) Burm, f. cv. Interdonato) seedlings. 6-Benzyladenine (BA) alone (1, 2 and 4 mg l⁻¹) and in combination with either orange juice (10%, v/v), silver nitrate (3 mg l⁻¹), gibberellic acid (GA₃) (0.1 mg l⁻¹ at the establishment stage and 0.5 mg l⁻¹ at all combinations during the proliferation stage) or abscisic acid (ABA) (0.2 mg l⁻¹ only at the establishment stage) were used to stimulate shoot formation during the establishment and the proliferation stage. The combination of BA with ABA gave a high rate of shoot formation, while GA₃ and silver nitrate enhanced shoot elongation. When these shoots were transferred to the rooting stage, the effect of application of two different auxins (indole-3-butyric acid (IBA) and α-napthaleneacetic acid) was examined, as well as different methods of application (auxin added to the basal medium and auxin application by dipping the base of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol solution of IBA at 1000 mg l⁻¹ for 5 s resulted in 80% rooting with subsequent 90% survival of these explants, during acclimatization under mist.     

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.     

Daylength and photon flux density influence the growth regulator effects on morphogenesis in epicotyl segments of Troyer citrange

The optimal growth regulator addenda for adventitious shoot regeneration in epicotyl cuttings of Troyer citrange (Citrus sinensis×Poncirus trifoliata) varied with the conditions of illumination. The response to illumination and to growth regulators differed for the direct and the indirect (through callusing) pathways of regeneration. Shoot formation through the direct organogenic pathway decreased as the concentration of benzyladenine (BA) in the medium was increased in the range 2.2–22 μM, when the explants were incubated in the dark or under an 8 h daylength. For explants incubated under a 16 h daylength, the number of shoots formed increased with BA concentration. Optimal conditions of incubation for shoot formation through the direct pathway were either an 8 h daylength with a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA, or a 16 h daylength with a photon flux density of 37 μmol m⁻² s⁻¹ in the presence of 22 μM BA. Irrespective of the conditions of incubation, shoot formation through the indirect organogenic pathway was suppressed by the addition of 22 μM BA to the medium. Optimal conditions for shoot formation through this pathway were incubation under an 8 h daylength at a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA. At the optimal conditions indicated, the addition of the synthetic auxin naphtaleneacetic acid (0.54 μM) reduced shoot formation. Irrespective of the pathway of regeneration, the number of shoots formed decreased markedly with the distance of the cutting from the cotyledonary node.     

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.     

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.     

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.     

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.     

Rapid in vitro multiplication and conservation of Garcinia indica: A tropical medicinal tree species

A simple and efficient method has been developed for rapid regeneration of plantlets via adventitious bud differentiation on mature seeds of Garcinia indica (Thouars) Choisy, a medicinally important facultative apomictic tropical tree species. High frequency direct shoot proliferation was induced in seed segments cultured on Murashige and Skoog's medium supplemented with cytokinins (BAP, kinetin and TDZ) alone and in combination with auxin (NAA). Amongst the various combinations used, BAP proved to be the most effective. Multiple shoots formed within 4–5 weeks of culture. The shoot forming capacity of the seeds was influenced by the BAP concentration tested (5–50 μM) and optimal response was observed at different concentrations (12.5–50 μM) in different genotypes investigated. Significant differences were recorded in terms of percent response (27.78–100%) as well as average number of shoots per explant (3.49–57.67) among the four genotypes investigated. Elongation of the induced shoots was achieved on MS basal medium containing 0.2% activated charcoal. The induction medium had a profound effect on rate of bud elongation with shoots induced on lower concentrations of BAP showing as much as four-fold elongation within 4 weeks. Proliferating shoot cultures were established by repeatedly subculturing the shoot nodes on MS medium supplemented with 5 μM BAP. Maximum rooting (91.66%) occurred in shoots cultured on half-strength MS medium supplemented with 10 μM IBA. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 15–17 weeks. For in vitro conservation, the shoot cultures were maintained on medium supplemented with 0.5 μM BAP and the subculture duration could be enhanced up to maximum of 11 months.     

Culture method and photosynthetic photon flux affect photosynthesis,growth and survival of Limonium ‘Misty Blue’ in vitro

Microcuttings (shoots each with two leaves) of Limonium ‘Misty Blue’ were cultivated in vitro for 28 days under photoautotrophic (sucrose-free culture medium; CO₂ and photosynthetic photon flux (PPF) enriched conditions), photomixotrophic (medium with 30 g l⁻¹ sucrose; CO₂ and PPF enriched conditions) and heterotrophic (medium with 30 g l⁻¹ sucrose; CO₂ non-enriched conditions) methods. Several growth variables were measured during and at the end of cultivation: shoot fresh and dry weight, percentage of shoot dry matter, root fresh weight, number of leaves, leaf area, chlorophyll and sugar content of leaves, stomatal density and size, net photosynthetic rate (NPR) and percent survival of plantlets ex vitro. Plantlets grown in photoautotrophic and photomixotrophic methods had more leaves, high chlorophyll and sugar contents, high NPR, and showed high percent survival. However, these plantlets possessed less number of stomata per square millimeter. In contrast, the plantlets grown by the heterotrophic method showed decreased values of these growth variables except for the number of stomata per square millimeter. These results indicate that CO₂ enrichment for plantlets in vitro at a relatively high PPF would promote photosynthesis and hence growth of chlorophyllous explants/plantlets in vitro. The resulting plantlets were acclimatized better and sooner on ex vitro transplantation.     

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.     

Micropropagation of Karonda (Carissa carandas) through shoot multiplication

Shoot tips from field grown, mature plants of Carissa carandas cv. Pant Sudarshan were cultured on Murashige and Skoog’s (MS) basal medium supplemented with benzyladenine (BA) and indolebutyric acid (IBA) during different seasons. The maximum sprouting rate was obtained with 1.5 cm long explant collected in spring season (February–March) followed by those collected in summer season (April–June). Shoot proliferation was highest on MS basal media supplemented with 3.0 mg l⁻¹ BA. Rooting of microshoots was noted to be the best in 1/2 MS plus 0.8 mg l⁻¹ IBA and 0.2 mg l⁻¹ naphthalene acetic acid (NAA). The rooted plantlets were successfully acclimatized in vermiculite:sand:soil (1:1:1) potting mixture.     

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 the shoot multiplication of Persian walnut (Juglans regia L.)

A number of experiments were conducted to identify suitable procedure for in vitro shoot multiplication of Persian walnut (Juglans regia L.). Three different nutrient media (DKW, MS and WPM) and three different gelling agents (Phytagel, Difco Bacto agar and a mixture of Phytagel and Difco Bacto agar) were studied in the first experiment. Driver and Kuniyuki walnut (DKW) medium solidified with 2.2 g l⁻¹ Phytagel was found optimum. Performance of explants was better on DKW medium than on MS and WPM. The DKW and MS media were not significantly different from each other, but both of them were significantly better than WPM, which was a very poor medium for this species. Phytagel alone was significantly better than Difco Bacto agar or Phytagel combined with Difco Bacto agar. In another experiment different concentrations of BA were studied. Medium containing 1.0 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was the best, although medium containing 0.6 and 0.8 mg l⁻¹ BA were also successful, and utilisation of 0.4 mg l⁻¹ BA and 0.01 mg l⁻¹ IBA was optimum for shoot elongation. Application of different kinds of auxins (IAA, IBA and NAA at 0.01 or 0.1 mg l⁻¹) with 1.0 mg l⁻¹ BA were also studied. Media containing IBA were significantly better than media containing IAA for shoot fresh weight, but neither of them was significantly different from media containing NAA. Application of 0.01 mg l⁻¹ or 0.1 mg l⁻¹ auxin, with 1.0 mg l⁻¹ BA, was not significantly different for shoot multiplication of Persian walnut. The morphology of shoots on media containing 0.01 mg l⁻¹ IBA was the best.     

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.