Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

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.     

Embryogenic callus formation from maize protoplasts

Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA fluorescein diacetate - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.     

Fertile indica and japonica rice plants regenerated from protoplasts isolated from embryogenic haploid suspension cultures

Summary A system to regenerate fertile rice (Oryza sativa L.) plants (both indica and japonica varieties) from protoplasts isolated from anther-derived embryogenic haploid suspension cultures has been established. Green plants were regenerated from protoplast-derived cell clusters five months after suspension culture initiation. Protoplast yields and subsequent growth of the protoplast-derived microcalli were enhanced by transferring suspension cells into AA medium (Muller et al. 1978) three to four days prior to protoplast isolation. Protoplasts were cultured initially in Kao medium (Kao et al. 1977) and in association with nurse cells for four weeks. Protoplast-derived microcalli were transferred onto N6 (Chu et al. 1975) or MS (Murashige and Skoog 1962) media for callus proliferation. Callus growth was more rapid and the calli were more enbryogenic when grown on N6 medium. The 2,4-D concentration used to develop the suspension culture was important. Cell cultures grown in medium containing 0.5 mg/l 2,4-D released protoplasts whose plating efficiency was higher than for protoplasts obtained from suspension cultures grown in 2.0 mg/l 2,4-D. However, suspension cells grown in 2.0 mg/l 2,4-D were superior with regard to the ability of protoplast-derived calli to regenerate green plants. Amongst several hormone treatments evaluated, a combination of 0.5 mg/l NAA + 5.0 mg/l BAP resulted in the largest number of green plants regenerated. There were no significant differences between BAP or kinetin regarding total number of plants regenerated. More than 200 green plants have been produced form six independently initiated suspension cell lines. The number of regenerated plants per 10⁶ protoplats plated anged from 0.4 to 20.0, and the average seed fertility of single panicles of these R_O plants was about 40%.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×10⁶ and 1×10⁷ protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×10⁵ / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.Abbreviations CMS Murashige & Skoog (1962) medium for cucumber - GLC gel-like callus - ESC established embryogenic suspension culture - 2,4-d 2,4-dichlorophenoxyacetic acid     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

Efficient plant regeneration from Indica (group 1) rice protoplasts of one advanced breeding line and three varieties

Regenerable embryogenic suspensions were established from one Indica (group 1) rice advanced breeding line and 9 Indica (group 1) rice varieties in 6–8 weeks. Four were chosen for protoplast culture and plant regeneration. About 4–7×10⁷ protoplasts were isolated from one gram of 8-week-old cell suspension. High plating efficiency (30.5%) and colony formation (13.7%) were obtained using nurse culture methods. A high plant regeneration frequency (67.5%) was observed for line IR57311-95-2-3. In total, 322 plants were regenerated. All the regenerated plants were fertile.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - NAA 1-naphtalene acetic acid     

Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza sativa L.)

Protoplasts were isolated from embryogenic cell suspensions obtained from mature seed derived embryogenic callus of the advanced Indica type rice breeding line IR72 available from IRRI (International Rice Research Institute), Manila. Culture of protoplasts with the agarose bead type method without nurse culture led to sustained proliferation of protoplast derived clones. A simple culture protocol was developed which stimulated embryogenic development. Germination of somatic embryos has so far produced 277 green plants from 6 independent experiments. 117 plants have been transferred to soil and are growing in the greenhouse. A few of them have already flowered and set seeds.Abbreviations 2,4-D, 2,4 Dichlorophenoxyacetic acid - BAP Benzylaminopurine - CH Casein hydrolysate - ECS Embryogenie cell suspension - Kn Kinetin - NAA Napthaleneacetic acid; Media: - AA Muller and Grafe 1978 - MS Murashige and Skoog 1962 - N6 Chu et al. 1975 - R2 Ohira et al. 1973     

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Summary Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Müller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.     

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5–12 months old cell suspensions showing visible colony formation in the range of 180–1050 colonies/10⁶ protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10⁶ protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - NAA naphthaleneacetic acid     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Plant regeneration from highly embryogenic callus,cell suspension and protoplast cultures of Trifolium fragiferum

Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum (2n=16). (L.). The best media for induction of embryogenic cultures were based on Kao (1977) or Kao and Michayluk (1975). Somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants, somatic embryo protoplasts and cell suspension protoplasts, leaflets and various explants of immature zygotic embryos. The process of somatic embryogenesis was maintained for over two years on Murashige and Skoog's (1962) medium supplemented with 0.5 mg l^-1 benzyladenine and 0.05 mg l^-1 naphthaleneacetic acid. These long term cultures were capable of regenerating plants that were fertile and produced seeds. These results were compared with those from protoplast, tissue and organ culture of other species of the Trifolium genus. This revised version was published online in June 2006 with corrections to the Cover Date.