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 from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genotypic variability for protoplast regeneration in Saintpaulia ionantha (H. Wendl.)

Summary The behaviour of eleven Saintpaulia ionantha (H. Wendl.) genotypes in protoplast culture was compared. Isolation of protoplasts from young shootlets regenerated in vitro on leaf explants, yielded 0.7 to 1.8 × 10⁶ protoplasts per gram fresh weight. In all cultivars and breeding lines tested, cell divisions were observed. The mean division frequencies varied between 1.0 and 5.0% after 14 days, and between 6.4 and 13.8% after 24 days of culture. In ten genotypes callussing and shoot regeneration were achieved. The difference between the genotypes in shoot regeneration rate, between 2 and 68%, was more pronounced. The comparison of four cytokinins indicated hat thidiazuron was most effective for shoot regeneration, but often resulted in poorer shoot quality than benzylaminopurine.Abbreviations BAP (6-Benzylaminopurine) - IAA (In-dole-3-acetic acid) - NAA (-Naphthaleneacetic acid) - TDZ (Thidiazuron = 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea) - 2iP (6-(,-dimethylallylamino)-purine)     

Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. ‘Lovely Tokyo’ and P. obconica cv. ‘Aalsmeer Giant White’. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.     

Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.)

A procedure is described to regenerate plants from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Somatic embryogenic calli were induced from ginger shoot tips on solid MS medium with half the concentration of NH₄NO₃ and supplemented with 1.0 mg l⁻¹ 2,4-Dichloroacetic acid (2, 4-D) and 0.2 mg l⁻¹ Kin. Rapid-growing and well-dispersed suspension cultures were established by subculturing the embryogenic calli in the same liquid medium. Protoplasts were isolated from embryogenic suspensions with an enzyme solution composed of 4.0 mg l⁻¹ cellulase, 1.0 mg l⁻¹ macerozyme, 0.1 mg l⁻¹ pectolyase, 11% mannitol, 0.5% CaCl₂ and 0.1% 2-(N-morpholino) ethane sulphonic acid (MES) for 12–14 h at 27°C with a yield of 6.27 × 10⁶ protoplasts g⁻¹ fresh weight. The protoplasts were cultured initially in liquid MS medium with 1.0 mg l⁻¹ 2, 4-D and 0.2 mg l⁻¹ Kin. Then the protoplast-derived calli (1.5 cm²) were transferred to a basal MS medium containing 0.2 mg l⁻¹ 2, 4-D, 5.0 mg l⁻¹ benzyladenine (BA), 3% sucrose and 0.7% agar. The white somatic embryos were transferred to MS medium lacking growth regulators for shoot development. Shoots developed into complete plantlets on a solid MS medium supplemented with 2.0 mg l⁻¹ BA and 0.6 mg l⁻¹ α-Naphthaleneacetic acid (NAA). In addition, the effects of AgNO₃, activated charcoal (AC) and ascorbic acid (AA) on browning of protoplast-derived calli are discussed.     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.     

Plant regeneration from haploid cell suspension-derived protoplasts of Mediterranean rice (Oryza sativa L. cv. Miara)

More than 750 plants were regenerated from protoplasts isolated from microspore callus-derived cell suspensions of the Mediterranean japonica rice Miara, using a nurse-feeder technique and N6-based culture medium. The mean plating efficiency and the mean regeneration ability of the protocalluses were 0.5% and 49% respectively. Flow cytometric evaluation of the DNA contents of 7 month old-cell and protoplast suspensions showed that they were still haploid. Contrastingly, the DNA contents of leaf cell nuclei of the regenerated protoclones ranged from 1C to 5C including 60% 2C plants. This was consistent with the morphological type and the fertility of the mature plants. These results and the absence of chimeric plants suggest that polyploidization occurred during the early phase of protoplast culture.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA3 gibberellic acid - NAA -naphthaleneacetic acid - PAS periodic acid Schiff - PCM protoplast culture medium - PCV packed cell volume     

Regeneration of shoots from cell suspension-derived protoplasts ofAllium cepa

Callus from pre-selected regenerating lines ofAllium cepa andA. fistulosum were used to initiate cell suspensions. Small clusters of callus selected for greater friability were placed into BDS liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), and were subcultured biweekly. Rapidly growing, finely dispersed lines were used for protoplast isolation. The highest yields came from 3–4 month old cell suspension lines. Protoplasts were cultured in modified K8P liquid medium. Microcalli recovery depended on the number of weeks the cell suspension had been in culture with highest recovery from 4–5 month old cell suspensions. Microcalli were moved to semisolid media when they were approximately 2 mm in diameter. After 4–6 weeks, embryogenic calli thus recovered were moved to variations of standard onion regeneration media containing picloram and BA. Elongating shoots were obtained from up to 88 % of the microcalli of one line, and 40–50 % of the shoots were further multiplied in culture.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - BDS modified B5 medium of Dunstan and Short (1977a) - K8P medium of Kao and Michayluk (1975) - MES 2-(Nmorpholino)ethanesulfonic acid     

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)     

Structure and Development of Plastids in Epidermal Cells of African Violet (Saintpaulia ionantha Wendl.) in Culture

The structure of plastids in epidermal cells of African violet(Saintpaulia ionantha Wendl.) ‘Marge Winters’ wasexamined using transmission electron microscopy before and afterplacement of leaf tissue in culture. The plastids from matureepidermal cells contained membrane-bound inclusion bodies andprolamellar bodies in various stages of development. Starchgrains and poorly developed granal stacks were observed in asmall number of plastids. After placement of the leaves on suitableculture medium, inclusion bodies decreased in size and the lamellarsystem became more organized. The plastids in the epidermaltissue are believed to be in an arrested state of developmentand are released from this state by placement on a medium inductiveto organogenesis. Saintpaulia ionantha Wendl., African violet, membrane-bound inclusion, tissue culture, plastid development     

Plant regeneration from leaf explants of Glycine clandestina Wendl

Fully fertile plants with the expected chromosome number 2n=40 were regenerated from excised leaf sections of Glycine clandestina. Shoots formed directly on the explants through organogenesis. Utilizing the same media and procedures fully fertile plants were also regenerated from cotyledon and hypocotyl tissue of the same G. clandestina accession. We were not successful in regenerating plants from root tissue of G. clandestina.Abbreviations 6-BA 6-Benzyladenine - FAA Formalin - NAA Naphthalenacetic acid - IAA Indoleacetic acid - Na₂EDTA Disodium salt Ethylenediamine tetraacetic acid - Fe-NaEDTA Ferric-Sodium salt Ethylenediamine tetraacetic acid     

Plant regeneration from root protoplasts of Brassica

Protoplasts isolated enzymatically from roots of Brassica alba (White Mustard), B. campestris (Turnip), B. napus (Rape) and B. oleracea (Cabbage), divided to form callus. Plant regeneration was obtained from protoplast derived tissues of B. napus and B. oleracea, but only rhizogenesis was observed with B. campestris. Tissues of B. alba remained undifferentiated. The suitability of root protoplasts for genetic manipulations in the genus Brassica is discussed.     

Plant regeneration from protoplasts of Capsicum annuum

Leaf protoplasts were isolated from axenic shoot cultures of four varieties of Capsicum annuum (Americano, Dulce Italiano Florida Gynat and Nigrum) and a wild species C. chinense. Protoplasts of both species, cultured in KM8P medium and using agarose bead culture, entered division with the exception of the variety Nigrum. Cell colonies formed callus in agar-solified MS medium supplemented with zeatin and for C. annuum v. Dulce Italiano shoots were regenerated when protoplast-derived calli were transferred to MS medium with 6-BAP. Excised shoots were rooted on MS medium which lacked phytohormones.Abbreviations 6-BAP 6-benzylamino purine - MS Murashige and Skoog (1962) - KM8P Kao and Michayluk (1975) - SDS sodium dodecyl sulphate     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid