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 meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

Microprojectile bombardment-mediated transformation of Lilium longiflorum

We have obtained transgenic lily (Lilium longiflorum) plants after microprojectile bombardment, using the Biolistics PDS 1000/He system, of morphogenic calli derived from bulblet scales, followed by bialaphos selection. Parameters which gave the highest transient uidA expression were used: a bombardment pressure of 1100 psi, a target distance of 6 cm and a 48-h preculture on medium with 3% sucrose. A total of 1800 morphogenic calli were co-bombarded with plasmids containing either the uidA reporter or PAT selectable marker genes. After bombardment, the calli were exposed to 2 mg/l bialaphos. Only 72 of the shoot-forming calli (4%) survived. The 72 shoot clusters produced 342 shoots on elongation medium containing 0.5 mg/l bialaphos. Only 55 plantlets survived subsequent exposure to 2.0 mg/l bialaphos. PCR analysis indicated that 19 of these plantlets contained the PAT transgene. Southern analysis of 3 of the plants indicated that all contained the PAT gene. Received: 21 March 1997 / Revision received: 8 July 1997 / Accepted: 7 August 1997     

An extensive hairy root production precedes shoot regeneration in protoplast-derived calli of cauliflower (Brassica oleracea var. botrytis)

Summary Protoplasts isolated from mesophyll tissues of cauliflower (Brassica oleraceavar. botrytis) were induced to divide in culture, with 2% of them producing calli. Upon transfer to a regeneration medium containing a low auxin/cytokinin balance (0.02mg/l 1-naphthaleneacetic and 2mg/l 6-benzylaminopurine), they displayed an extensive production of hairy roots before the regeneration of shoots. Negative effects of root differentiation on the subsequent caulogenesis by such calli were not observed, since 97% of the calli regenerated hairy roots and 93% gave shoots.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - MES 2-N-morpholinoethane sulfonate     

In Vitro Culture and Regeneration of Solanum khasianum and Extraction of Solasodine

Leaf explants of Solanum khasianum regenerated on MS medium containing 2, 4-D (3 mg/l) and kinetin (1 mg/l). Shoots could be induced from these calli on medium containing BAP (3 mg/l) alone. Rhizogenesis of these shoots occurred when transferred to medium containing 2 mg/l NAA. The yield of solasodine — a pharmaceutically important compound, from 4-month-old callus tissue was remarkable at 2 per cent of dry weight.     

Somatic embryogenesis and plant regeneration in callus culture of tef, Eragrostis tef (Zucc.) Trotter

The study was carried out to establish in vitro culture conditions for plant regeneration of tef, Eragrostis tef (Zucc.) Trotter. Mature seeds of two Ethiopian varieties, DZ-01-354 and DZ-01-196, were used to initiate callus cultures on Murashige and Skoog (MS) medium with different auxins. Four- and 8-week-old calli induced on a medium with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were subcultured onto various media to induce somatic embryogenesis. Compact, nodulated, embryogenic callus was observed after transfer onto MS-callus proliferating (CP) medium. Embryogenic tissue appeared on soft and amorphous callus and developed into somatic embryos during a subsequent subculture to MS embryo-promoting (EP) media. Various growth regulator combinations were tested in CP and EP media to obtain a high efficiency of somatic embryo formation. The highest frequency of calli forming somatic embryos (56.1–68.3%) was observed when CP media with 2.0 or 4.0 mg/l 2,3,5-triiodobenzoic acid were employed and then cultures were transferred to EP media with 0.5 mg/l 2,4-D and 0.5 mg/l kinetin followed by 0.5 mg/l indole-3-acetic acid and 0.5 mg/l N⁶-benzyladenine. Plant development from somatic embryos was obtained on MS medium supplemented with 1.0 mg/l gibberellic acid. On average, 71.2% of calli displaying somatic embryos converted into plants. Regenerated plants were successfully transferred to soil. Neither chlorophyll-deficient plants nor morphological variants were found among regenerants. All regenerated plants were fertile. Received: 9 May 1997 / Revision received: 25 September 1997 / Accepted: 3 January 1998     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

The Effect of High-Concentration Auxin and Plant Regeneration in the Immature Embryo Culture of Soybean

Using immature embryos of soybean as explants, green structures and somatic embryoids were able to be induced on higher auxin-containing media. Genotypes, developmental degree of the embryos, origin of the explants and medium compositions all affected the occurrence of the structures and calli. After the green structures were transferred to high 2,4-D containing medium (30 mg/l) calli were reinduced. These calli were maintained on the same medium without being subcultured for 2 months and then transferred to lower hormone-containing media. After 2 weeks, a great number of new green structures in the same shapes were induced. It was shown that high level of 2,4-D played a unique role in lasting the morphogenesis ability of the cultures. When the green structure were cultured on low hormone-containing media they developed new leaves and formed leaf clusters while the apical did not develop. In order to stimulate the apical development the medium containing 2 mg/l GA3 and 0.1 mg/l IBA was used and some plantlets were obtained. The different effects of NAA and 2,4-D on the explants and calli were studied. Calli induced from the cotyledon of immature seeds (416 mm) had a regeneration ability stronger than that from the seedlings. The calli induced by use of the medium containing high concentration of 2,4-D (5–30 mg/l) have higher potentialities in producing green structures. In contrast, the calli induced by high concentration of NAA (10 mg/l) were highly root-morphogenetic. The explants and the calli cultured on the medium containing 5 mg/l 2,4-D could be maintained for a long term without being subcultured frequently.     

An efficient regeneration system and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese upland rice cultivar Handao297

A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants. Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots by five to tenfold in comparison to medium without TDZ.     

Effect of basal medium, growth regulators and Phytagel concentration on initiation of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.)

Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM₁ basal medium proved superior to DCR₁ and LP (LP basal salts plus BM₁ organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 September 1997     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Genetic polymorphism analysis of somatic embryo-derived plantlets of Cymbopogon flexuosus through RAPD assay

The genetic status of somatic embryo-derived plantlets of Cymbopogon flexuosus was examined by randomly amplified polymorphic DNA (RAPD) analysis. Auxins such as 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1–4 mg/l) were used in Murashige and Skoog (MS) medium for induction of calli from rhizomatous explants of Cymbopogon flexuosus. Optimum calli were induced on MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) (3.5 mg/l) alone or in combination with N ⁶-benzyladenine (2 mg/l). Somatic embryogenesis was achieved from long term calli when cultured on MS medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D) (2 mg/l) along with N ⁶-benzyladenine (BA) (1–2 mg/l). Regeneration was achieved when freshly induced embryogenic calli were sub-cultured on MS medium supplemented with N ⁶-benzyladenine (3 mg/l) alone. Long-term cultured embryos showed profuse minute rooting on regeneration medium supplemented with N ⁶-benzyladenine (3 mg/l). Microshoots were rooted in the presence of indole-butyric acid (IBA) (2 mg/l). DNA samples from the mother plant and 18 randomly selected regenerated plants from a single callus were subjected to RAPD analysis with 6 arbitrary decamer primers for the selection of putative somaclones. A total of 64 band positions were scored, out of which 19 RAPD bands were polymorphic. From genetic similarity coefficient based on RAPD band data sharing, it was found that the majority of the clones were almost identical or more than 92% similar to the mother plant, except CL2 and CL9 (66%) which showed highest degree of genetic change with CL2 and CL9 showing presence of two non-parental bands each.     

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Production of plantlets from Aegle marmelos nucellar callus

Techniques have been developed for the regeneration of Aegle marmelos from nucellar explants. Slow-growing calli were induced from nucellar explants excised from 90–120 d-old developing fruits. The medium consisted of Murashige and Skoog formulation containing 40 g/l sucrose, 400 mg/l casein hydrolysate, 5 mg/l 1-naphthaleneacetic acid and 1 mg/l kinetin. The basal medium with high concentration (1–5 mg/l) of N6-benzyladenine (BA) and low concentration (0.1 mg/l) of NAA was suitable for regeneration of shoots from 3-month-old calli. Addition of 1 mg/l gibberellic acid (GA3) favoured shoot growth. Callus-derived shoots produced roots and developed into plantlets when transferred to half-strength MS medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 0.5 mg/l NAA. Approximately 5 months were required for the full regenerative process.     

Organogenesis and Plantlet Formation from Organ- and Seedling-Derived Calli of Rice (Oryza sativa)

Callus was induced in different somatic organs of Oryza sativa L. Specific minimum 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations in the medium were necessary for the induction of callus from different organs while high levels of 2,4-D (6–10 mg/l) induced callus formation in each organ tested. The optimum 2,4-D concentration for callus induction and growth for root-derived calli was 2 mg/l and for leaf-derived 6 mg/l. Root and shoot organogenesis were induced in both root- and leaf-derived calli by sub-culturing to a medium lacking 2,4-D. Root organogenesis occurred at a higher frequency than shoot organogenesis. Shoot organogenesis rarely occurred in calli without differentiated roots. Increased age of callus cultures almost completely inhibited shoot development. The addition of the cytokinin 6-γ,γ-dimethylallyl-amino purine partially restored the potential for shoot organogenesis. Whole plants were easily recovered from the calli and grown to maturity with some plants exhibiting phenotypic abnormalities.     

Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria

 Stem segments of seedlings from two Alstroemeria breeding lines, cultured on media supplemented with 4 mg/l 2,4-dichlorophenoxyacetic acid and 0.5–1.0 mg/l 6-benzylaminopurine (BA), initiated soft callus, which became compact after subculture on a medium with only 0.5 mg/l BA. Friable embryogenic calli were initiated from compact callus on a medium supplemented with 10 mg/l picloram. Proembryos developed from friable embryogenic calli via embryos into plants after subculture on medium supplemented with 0.1 mg/l BA. The proembryos formed friable embryogenic calli again after culture on medium supplemented with 10 mg/l picloram. The total time needed to regenerate a complete plantlet from friable callus was approximately 6 months. This system for the production of embryogenic material is considered to have valuable applications for genetic transformation in Alstroemeria. Received: 22 April 1999 / Revision received: 16 July 1999 · Accepted: 20 July 1999     

Organogenesis and plant regeneration in Zephyranthes rosea Lindl.: Histological and chromosomal study

The genus Zephyranthes rosea is a member of the family Amaryllidaceae. The plant is widely cultivated as ornamental. The objective of this study was to optimize an in vitro propagation method for the production of genetically stable Z. rosea plant. The chromosomal status of the regenerated plants was also studied to determine their ploidy levels and to identify the structural and numerical variations, if any. Two explants of Zephyranthes rosea, i.e. bulb scale and flower bud (3–4 mm each), were used and incubated in a culture room at 25 ± 2°C in which two different types of calli were induced from two sources. The MS medium amended with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5–2.0 mg/l) successfully induced callus from bulb-scale explants (50.25–57.5%). The addition of coconut water (10%) in 2,4-D-added medium further improved the callus induction frequency (68.4%). Bulb-scale calli were found to be highly regenerative while flower-bud calli did not show any organogenetic responses. The use of plant growth regulators, such as naphthaleneacetic acid (NAA) + benzylaminopurine (BAP), was found to be very effective for shoot bud development; maximum shoot number (11.50/callus mass) was observed in NAA (0.5 mg/l) + BAP (1.0 mg/l) added medium. Histological analysis of callus revealed that the origin of the shoot bud was de novo. Rooting frequency (65.25%) and the number of roots (7.5/shoot) were best achieved in indole-3-butyric acid (4.0 mg/l)-amended medium, followed by indole-3-acetic acid (4.0 mg/l). The regenerated Z. rosea plants showed 2n = 24 chromosome numbers.     

Somatic embryogenesis from integument (perisperm) cultures of coffee

Somatic embryogenesis was induced in integument (perisperm) cultures of C x R hybrid cultivar of coffee, after a culture period of 15 months, using a sequence of 3 modifications of MS medium. Vigorously growing soft, white, watery crystalline calli were obtained on MS + TIBA (1 mg/l) + L-cysteine HCl (50 mg/l) + PVP (100 mg/l). After 45 d, the calli were subcultured to MS + IAA (0.5 mg/l) + 2,4-D (0.05 mg/l) + Kn (8.6 mg/l) and maintained for the next 9 months without any transfer. On this medium, the callus proliferation was initially vigorous which slowed down after 5–6 months, and then the calli turned light brown and somewhat compact. Later, when the calli were transferred to MS + thiamine HCl (10 mg/l) + pyridoxine HCl (3 mg/l) + nicotinic acid (2 mg/l) + 2,4-D (0.2 mg/l) + 2ip (2.5 mg/l) and cultured for 2 months, they turned darker, more compact and the proliferation almost stopped. These calli were subcultured onto fresh medium of the same composition. After another 2 months of culture cream-coloured, highly friable, embryogenic calli appeared, which in turn produced a few clearly identifiable SEs in another 1 month. Further proliferation and maturation of SEs was achieved by culturing the embryogenic calli on MS + ABA (1 mg/l) for 3 months. The SEs were germinated into 2 cm tall plantlets after 2–3 subcultures, each of 2 months duration on 1/2-MS + Kn (0.1 mg/l).Abbreviations MS Murashige and Skoog (1962) basal medium - ABA Abscisic acid - TIBA 2,3,5 -Triiodobenzoic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-dichloro-phenoxyacetic acid - Kn Kinetin - 2ip N⁶-(2-isopentenyl) adenine - PVP Polyvinylpyrrolidone; - SEs Somatic embryos     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri

This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7 mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt solution with 7 mm CaCl₂ ^.2H₂O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks. Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997