Mesophyll protoplasts from Solanum melongena var depressum bailey regenerate to fertile plants

Mesophyll protoplasts have been isolated from soil grown plants of Solanum melongena var depressum Bailey and protoplast-derived colonies regenerated in liquid DPD culture medium. Protoplast-derived colonies proliferated on agar-solidified MS culture medium and regenerated shoots which grew to fertile plants in potting compost.on leave from Landzhou University, Peoples Republic of China     

Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.)

Summary Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l^-1 6-benzyl-aminopurine and 0.1 mg.l^-1 l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - IBA 4-indole-3yl-butyric acid - IPE initial plating efficiency - f wt fresh weight - KM Kao and Michayluk (1975) - MES 2-N-morpholino ethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA l-naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000)     

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     

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     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea 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%.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Cauliflower inflorescence protoplast culture and plant regeneration

A yield of 2–4×10⁶ protoplasts/g F.W. could be obtained when fresh cauliflower inflorescence segments were digested with 2% cellulase Onozuka R-10, 1% cellulase RS and 0.4% Macerozyme R-10 in CPW18S for 7 to 10 h. Purified protoplasts were cultured in K8p liquid and agarose medium. Although protoplasts in liquid medium divided earlier than in agarose, protoplast-derived cells in liquid culture could not avoid browning. With agarose culture, sustained division and callus formation could be achieved. After 20 days, calli were transferred onto B5 agar medium with ZT 1.5 mg l^-1, BA 0.5 mg l^-1 and IAA 0.1 mg l^-1 for shoot formation. The frequency of bud formation varied from 56.7% for calli of 1mm in size to 5.6% for 5mm calli. The shoots formed were rooted in B5 medium containing 0.5 mg l^-1 IBA, and the regenerated plants were transplanted to pots and grew normally. It took about two months from protoplasts to the regenerated plants.Abbreviations Ade adenine - BA 6-benzyl aminopurine - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4,-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Gln glutamine - NAA -naphthylacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - ZT zeatin     

Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.)

Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T₃) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants.Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplastderived calli from Fujiminori, produced whole plants in the regeneration medium.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

Isolation,culture, and regeneration of plants from potato protoplasts

A technique is described for the routine isolation of protoplasts from storage parenchyma cells of potato tubers grown in vitro. The protoplasts typically contained many starch grains. On culture, most of the starch grains were metabolised during the first 7 days, after which the cells began to divide. Following further culture, protoplast-derived colonies and calli were obtained, from which shoots and intact plants were regenerated. Cytological study of regenerated plants showed that the majority were octaploid or aneuploid at the octaploid level. This aspect is compared with plants regenerated from mesophyll protoplasts of potato. The use of tuber protoplasts for studies on tissue-specific transient gene expression of chimeric gene constructs, following their introduction into the protoplasts by electroporation, is discussed, together with the uses of tuber protoplasts in fundamental physiological and biochemical studies.     

Plant regeneration from cultured cell-derived protoplasts of Pelargonium aridum,P. x hortorum and P. peltatum

Cultured protoplasts from cell suspensions of Pelargonium aridum, P.x hortorum and P. peltatum divided and formed callus. On agar-solidified regenerative medium, such protoplast-derived calli (p-calli) underwent plant regeneration at frequencies approaching 100% for P. aridum and 10% for P.x hortorum. Under similar conditions shoot primordia arose in 5% of P. peltatum p-calli, but these never developed into normal shoots. However, following a liquid-shake culture regime, whole plants were induced in 20% of P. peltatum p-calli. This approach also improved regeneration of P.x hortorum to 60%.Abbreviations NAA napthaleneacetic acid - 6-BAP 6-benzylaminopurine     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

Plant regeneration from protoplasts of hydroxyproline resistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onobrychis viciaefolia was established.In SH medium supplemented with 1mg/L2,4-dichlorophenoxy-acetic acid(2,4-D),0.5mg/L kinetin(KT)and 0.2mg/L naphthalene acetic acid(NAA),the division frequency of protoplastderived cells reached up to over 60%,and microcalli were obtained in 5-6wk.Upon transferring them on agar solidified MS medium plus 2mg/L indole-3-acetic acid (IAA),shoots were induced.After cultivating them on MS medium with or without IAA,roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal(2n=28).The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.     

Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L.)

A procedure is described for the regeneration of plants from protoplasts of tetraploid leek (Allium ampeloprasum L.), 2n = 4x =32. Regeneration-competent protoplasts could only be obtained from an embryogenic suspension culture that was initiated with friable, embryogenic callus derived from immature embryos. The generally low plating efficiency could be increased by embedding the protoplasts in Ca-alginate, compared to culturing the protoplasts in liquid or agarose-solidified medium. A minimum plating density of 2 × 10⁵ pps/ml was required to obtain microcalli. Upon transfer of the protoplast-derived calli on agarose-solidified BDS medium, morphologically different callus types proliferated. After transfer to regeneration medium, compact or friable calli with an embryogenic appearance produced somatic embryos and plantlets at a frequency of up to 80%. Calli that had been classified as heterogeneous also regenerated shoots, but mainly via organogenesis, at a frequency of 46%. After transfer of shoots to half strength MS medium, healthy, well-rooted plants were obtained, that were successfully transferred to soil. All plants contained the tetraploid DNA level.     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid