Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium × formolongi by utilizing meristematic nodular cell clumps

Plant regeneration from protoplasts of Lilium × formolongi cv. Azusa was achieved by utilizing suspension cultures of meristematic nodular cell clumps with a high plant regeneration ability. Creamy-white calli with embryogenic potential were initially induced from the seeds on Murashige and Skoog (MS) medium containing 4.1 μM picloram. The calli were then transferred into a liquid medium of the same composition, in which they turned into compact cell clumps which consisted of meristematic nodules. Protoplasts were readily isolated from these meristematic nodular cell clumps. Colonies were successfully formed from the protoplasts by embedding in 0.1% gellan gum-solidified MS medium containing 4.1 μM picloram and 0.5 M glucose. They regenerated shoots and roots on MS medium containing 2.2 μM benzylaminopurine (BAP). The plants thus obtained produced flowers with normal fertile pollen 8 months after successful transfer into soil. These plants had normal chromosome numbers (2n = 24) but had shorter leaves than original plants. They set seeds after as well as cross pollination.     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant Regeneration from Protoplasts of Peucedanum Terebinthaceum (Fisch.) Fisch. ex Turcz

Calll with many embryogenic cell colonies were produced from segments of seedlling of Peucedanum terebinthaceum (Fisch.) Fisch. ex Turcz. which were cultured on the 1/2MS agar medium (with half quantity of macronutrients) containing 1 mg/l 2,4-D. Cell suspension culture with high percentage of embryogenic cell colonies was established from the calli shaking in liquid medium. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CeCl2, 1 mmol/l KH2PO4, 0.6 mol/l mannital at pH 5.8 and 25℃. Cultured in a modified MS liquid medium containing 1 mg/l 2,4-D+ 0.5 mg/l zeatin, the protoplasts emered division after four days, and formed cell colonies of 0.5–1mm after about forty days. When transfered to 1/2 MS liquid medium supplemented with zeatin (0.5 mg/l), the cell colonies differentiated in to embryoids, then developed into plantlets with many green leaves and roots on the 1/2 MS agar medium devoid of phytohormones.     

Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.)

Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×10⁶ protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - PCV packed cell volume - MES morpholinoethanesulfonic acid     

Rapid and efficient plant regeneration from hypocotyl protoplasts of Brassica carinata

Protoplasts were isolated from hypocotyls of 7-d-old seedlings of three genotypes of Brassica carinata after enzymatic digestion in cellulase R-10 (0.5%) and pectolyase Y-23 (0.025%). The protoplasts were stabilized with 0.4 M mannitol used as osmoticum, and were cultured in darkness in Kao's liquid medium containing 0.4 M glucose and the growth regulators 2,4-D (1.0 mg/l), NAA (0.1 mg/l) and zeatin riboside (0.5 mg/l). Protoplasts were transferred to 16 h photoperiod conditions after 3 d of dark culture, and the medium was diluted to reduce the osmoticum on the seventh and tenth days of culture. Microcolonies were thus obtained which, upon transfer to MS agarose medium with 2,4-D (0.1 mg/l), BAP (1 mg/l) and 0.1 M sucrose, proliferated further to produce callus clumps. The plating efficiency of the three genotypes varied from 1 to 2%. Calli 2–3 mm in diameter were transferred to MS agarose plates with zeatin (2 mg/l) where they produced shoot buds and shoots with frequencies ranging from 22.5 to 74.2% for the three genotypes. The shoots were rooted in medium with IBA (1 mg/l) and were then established in soil. The time required for protoplast to plant development was 8 to 10 weeks.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -naphthalene acetic acid - BAP 6-Benzylaminopurine - KN Kinetin - 2IP 6-(Gamma, gamma-dimethylallyl-amino)purine     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis

Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Plantlet Regeneration from Protoplasts lsolated from an Embryogenic Suspension Cultureof Cotton(Gossypium hirsutum L.)

Protoplasts were isolated from an embryogenic suspension culture of commercial cotton cv. The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium with K3 mineral salts and modified Km8p organic compositions, supplemented with 0.05–0.1 mg/l 2,4-D, 0.2–0.5 mg/l 2ip in the dark. The regenerated plantlets from protoplasts of coker312 and coker 201 cv. were obtained. Embryogenesis from protoplast of Jin4 cv. and microcolonies form protoplasts of JiHe321 and Lul cv. were observed.     

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA), or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.     

Somatic embryogenesis in Narcissus pseudonarcissus cvs. Golden Harvest and St. Keverne

Somatic embryos (SEs) have been produced from bulb and shoot culture leaf explants of Narcissus pseudonarcissus cvs. Golden Harvest and St. Keverne. Initial experiments with cv. Golden Harvest resulted in SEs from leaf lamina, leaf base, bulb scale and scape (flower stem) explants. Embryogenesis was induced on media with a range of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) concentrations. There were significantly more SEs with media containing 5 μM 2,4-D and 0.5 μM or 5 μM BAP than any other growth regulator combination. Scape explants produced more early SEs than the other explant types, and when orientated with their basipetal surface away from the medium, they produced significantly more advanced SEs than those with this surface in contact with the medium. Leaf explants from shoot cultures of cv. Golden Harvest produced SEs on medium with BAP combined with 2,4-D or 1-naphthaleneacetic acid (NAA), but 4-amino-3, 5, 6-trichloro-2-pyridinecarboxylic acid (picloram) was ineffective. SEs converted to plantlets efficiently following a 4°C treatment in addition to 4.9 μM indole-3-butyric acid (IBA). These plantlets readily transferred to ex vitro conditions.     

A simple culture method for Brassica hypototyl protoplasts

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2–4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4–6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3–4 weeks.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4 — dichlorophenoxyacetic acid     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

从石刁柏原生质体培养再生植株

石刁柏,又名芦笋(Asparagus officinalisL.)是百合科天门冬属植物。其栽培品种含有丰富的维生素类及蛋白质。同时,石刁柏对于某些疾病有一定的药效,因此它已成为人们所喜爱的一种高级营养蔬菜。国外已有不少关于石刁柏试管苗繁殖的报告,但至今只有Bui Dang Ha等从石刁柏枝状叶分离的原生质体得到愈伤组织,并由此愈伤组织诱导获得了再生植株。此后,未见在石刁柏的原生质体培养方面再有新的工作。在本文中,我们利     

Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 mg/l 2,4-D, 0.5 mg/l BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8±0.5% and 6.5±0.7%, respectively. Upon transfer to Murashige and Skoog (1962) medium (MS) with 1–2 mg/l BA, alone or in combination with NAA or 2,4-D at 0.1 mg/l, the protoplast-derived calli produced complete plantlets through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5± 2.2%) occurred on MS medium containing 0.1 mg/l NAA and 1 mg/l BA, whereas the maximum number of calli regenerating plantlets (64.7±6.2) was obtained on MS medium with 0.1 mg/l NAA and 2 mg/l BA. Received: 25 April 1997 / Revision received: August 1997 / Accepted: 2 September 1997     

Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var ‘capitata’)

Summary Protoplasts were enzymatically isolated from the first leaves of cabbage (Brassica oleracea var capitata, F1 hybrid Baochun). Sustained cell division and somatic embryogenesis were obtained after culturing the protoplasts in modified liquid DPD medium supplemented with CaCl₂ · 2H₂O 800 mg/l, 2,4-D 0.5 mg/l, kinetin 1 mg/l, 0.3 M mannitol and sucrose 20 g/l. Upon transferring cell colonies onto a modified Murashige and Skoog (MS) agar medium, small calli were gradually formed. Callus proliferated on MS medium supplemented with hormone combinations of 2,4-D 0.1–0.5 mg/l and kinetin 3–4 mg/l. Multiple shoots were induced on differentiation medium supplemented with 3 mg/l of kinetin and 0.1 mg/l of gibberellic acid GA₃. After transferring differentiated shoots onto MS medium supplemented with indoleacetic acid (IAA), kinetin, GA₃ at 0.1 mg/l each and 500 mg/l of N.Z. amine, intact plants were eventually produced.