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.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

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     

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 10⁵ protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.8 mg l⁻¹ 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.     

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     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Optimized Chemodiversity in Protoplast-derived Lines of St. John's Wort (<Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.)

Summary A procedure for protoplast isolation and plant regeneration of St. John's wort has been developed to utilize cell-to-cell variability for optimum production of valuable medicinal compounds. Calluses, induced from hypocotyl segments of St. John's wort seedlings, were used for protoplast isolation, induction of sustained cell division, and ultimately, plant regeneration. Callus-isolated protoplasts at a density of 2.0×10⁵ per ml were embedded in 0.6% Na-alginate blocks and cultured in a medium containing modified Murashige and Skoog (MS) salts, 2.5 μM 6-benzylaminopurine (BA), 5.0 μMα-naphthaleneacetic acid (NAA), and 0.5 moll⁻¹ glucose. Protoplast-derived colonies formed compact calluses when transferred onto 0.35% gellan gum-solidified MS medium supplemented with 2.5 μM BA and 2.5 μM NAA. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5 μM thidiazuron. Complete plantlets were obtained from the regenerated shoots on MS basal medium. A greater than 3-fold variation of antioxidant activity was observed among the protoplast-derived plantets and chemically distinct germplasm lines were selected on the basis of phytochemical profiles. The protoplast to plant regeneration protocol developed in this study provides the foundation for development of novel genotypes with potential expansion of the genetic diversity through somatic hybridization, and organelle transplantation.     

Plantlet regeneration from protoplast-derived haploid embryogenic calli of wheat

To search for an alternative method for protoplast culture, regenerable embryogenic calli were obtained from anther culture of three wheat cultivars, Karl 92, Jinghua #1, and Pavon 76. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in modified PMI and LM8P media without going through cell suspension culture. After 8–11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3–4 × 10⁵ protoplasts ml⁻¹. Frequency of colony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, while Karl 92 failed to produce colonies, even though its embryogenic calli were friable and fast-growing on the maintenance medium. Green haploid plantlets of Jinghua #1 and Pavon 76 have been regenerated from protoplasts, which were cultured on a differentiation medium first and then on a rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Efficient plant regeneration from cell suspension protoplasts of the woody medicinal plant Solanum dulcamara L. (bittersweet,woody nightshade)

Large populations of viable protoplasts were released from suspension cultured cells of the woody medicinal plant Solanum dulcamara (bittersweet, woody nightshade) when the cells were harvested 3 to 7 months after culture initiation and 4 to 5 days after transfer to fresh medium. A Bio-Gel p6 purified enzyme mixture enhanced the protoplast plating efficiency 6 fold compared to the unpurified mixture, without affecting protoplast yield. Agarose-solidified medium markedly improved protoplast division and colony formation, and enabled protoplasts to be plated at lower densities than in liquid medium. All protoplast-derived tissues produced shoots on MS based medium with 1.0 mgl^-1 zeatin. Shoots rooted readily on medium lacking phytohormones. Cytological examination revealed high chromosome stability of suspension cultured cells, of plants derived from such cells, and of protoplast-derived plants. The implication of these results is discussed in relation to the genetic manipulation of this pharmaceutically important plant.     

Cytoplasmic-nuclear interaction and medium effect for protoplast culture in sunflower

Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with a density of 2×10⁴ protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from 0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions. Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower. This revised version was published online in June 2006 with corrections to the Cover Date.