Plant regeneration from embryogenic cell suspension cultures of <Emphasis Type="Italic">Lolium temulentum</Emphasis>

Summary Lolium temulentum L. (Darnel ryegrass) is a self-fertile and diploid grass species with a relatively short life cycle. We propose to use L. temulentum as a model system for genetic manipulation studies in forage and turf grasses, since most of the important grasses are outcrossing, require vernalization to flower, and in some cases are polyploid. As the first step to develop an efficient regeneration and transformation system, we performed a large-scale genotype screening for tissue culture responses using 46 L. temulentum accessions. Embryogenic callus formation frequency ranged from <1% to 11% across all accessions tested. Embryogenic calluses of a few responsive accessions were used to establish cell suspension cultures. The regeneration frequency of green plantlets from the established cell suspension ranged from 15% to 39%. After transferring the regenerants to the greenhouse, fertile plants were readily obtained without any vernalization treatment. This efficient plant regeneration system is being used for genetic transformation studies. With the development of genomics approaches for the improvement of forage and turf grasses, L. temulentum could serve as a model system for testing gene functions.     

Plant regeneration from embryogenic cell suspension cultures of wild sorghum (Sorghum dimidiatum Stapf.)

A simple and efficient protocol is described for regeneration of wild sorghum (Sorghum dimidiatum) from cell suspension cultures. Fast-growing cell suspensions were established from shoot-meristem-derived callus. Plating of the suspension on Murashige and Skoog agar medium supplemented with 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in the formation of embryogenic calli. High-frequency (80%) somatic embryogenesis from small cell clusters (300–400 μm) was observed when the cultures were initially maintained in liquid medium with reduced levels of 2,4-D (0.25 mg l^–1), followed by transfer to regeneration medium. Direct plating of these small clusters on regeneration medium or transfer to liquid regeneration medium containing kinetin and 6-benzylaminopurine resulted in the development of mature somatic embryos and plantlets. The regenerants developed to maturity and were all phenotypically and cytologically normal. Received: 20 May 1998 / Revision received: 1 September 1998 / Accepted: 23 September 1998     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

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.     

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.     

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 10⁵/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.     

Plant regeneration from protoplasts isolated from cell suspension cultures of the wild tomato,Lycopersicon chilense Dun.

A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l^-1 zeatin and 0.1 mg l^-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.Abbreviations BAP 6-benzylaminopurine - IAA indole acetic acid - IBA indole butyric acid - MES-2 (N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

A cultivar comparison of plant regeneration from suspension cells,callus, and cormel slices ofGladiolus

Callus was initiated from either cormel slices or in vitro-grown plants of sixGladiolus cultivars cultured on Murashige and Skoog&#x2019;s basal salts medium supplemented with either 10 mg/liter (53.8 &#x00B5;M) 1-naphthaleneacetic acid, 2 mg/liter (9.3 &#x00B5;M) dicamba, or 0.5 mg/liter (2.2 &#x00B5;M) 2,4-dichlorophenoxyacetic acid. More plants were regenerated from callus of the cultivar &#x201C;Peter Pears&#x201D; as compared to &#x201C;Jenny Lee,&#x201D; &#x201C;Florida Flame,&#x201D; or &#x201C;Golden Year.&#x201D; No plants were regenerated from callus of &#x201C;Rosa Supreme&#x201D; or &#x201C;Purity White.&#x201D; Plants were regenerated from 2 and 6-mo.-old suspension cells of &#x201C;Jenny Lee&#x201D; and &#x201C;Peter Pears&#x201D; but not from &#x201C;Florida Flame.&#x201D; Cormel slices cultured on Murashige and Skoog&#x2019;s basal salts medium supplemented with 1 mg/liter (4.4 &#x00B5;M) 6-benzylaminopurine regenerated plants from all six cultivars indicating a cultivar-independent system of plant regeneration.     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

几种生理因子对印楝细胞悬浮培养生长的影响

通过研究不同生理因子对印楝悬浮细胞生长的影响 ,建立了一种快速生长的印楝细胞悬浮培养体系。结果表明 ,在添加了 6 BA 0 .8mg/L、NAA 0 .8mg/L、蔗糖 3 0 g/L且初始 pH值为 5 .8的MS培养液中 ,以 60g/L的接种量进行印楝细胞的悬浮培养 ,细胞生长速率可达 4.49g/L·d。     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L

Summary Embryogenic cell suspension cultures and somatic embryos of five genotypes of beech, were obtained from aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2, 4-dichlorophenoxyacetic acid and N6-benzyladenine. The origin of somatic embryos was traced from single cells. Embryos remained arrested at the globular stage on liquid media, further development was achieved after plating embryogenic aggregates on Murashige and Skoog's medium with half strength major salts supplemented with glutamine and low levels of growth regulators. Cultures of different genotypes showed significant differences in maturation frequency which was not affected by the hormone treatments assayed. The frequency of conversion of embryos into plantlets was low. This frequency increased after cold storage of embryos for up to 7 months.Abbreviations BA N6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOH ethanol - GA₃ giberrellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - WPM woody plant medium (Lloyd and McCown, 1980) - Z zeatin     

The differential effects of C-16,17-dihydro gibberellins and related compounds on stem elongation and flowering in Lolium temulentum

Plants of Lolium temulentum L. cv. Ceres grown under short days (SDs) can be induced to initiate inflorescences either by exposure to one long day (LD) or by single applications of some gibberellins (GAs), which also enhance the flowering response to one LD. Single doses of up to 25 μg per plant of C-16, 17-dihydro-GA₅ were about as effective as GA₅ for promoting flowering after one LD but inhibited stem elongation by up to 40% over three weeks. The promotion of flowering but not the inhibition of elongation by 16, 17-dihydro-GA₅ was reduced in SDs or in LDs low in far-red (FR) radiation. With shoot apices cultured in vitro, 16, 17-dihydro-GA₅ was more florigenic than GA₃ for apices excised after one LD of 14 h or more, but less florigenic for apices excised from plants in shorter days. 16, 17-Dihydro-GA₅ was ineffective compared with GA₁, GA₃ and GA₅ for α-amylase production by half-seeds of Lolium, a response concordant with its effect on stem elongation. As with GA₅, 16, 17-dihydro derivatives of GA₁, GA₃, GA₂₀ and several other GAs were more effective for flowering and less effective for stem elongation than the GAs from which they were derived. Hydroxylation at C-17 and/or C-16 generally reduced the effectiveness of 16, 17-dihydro-GA₅ for flowering. These results extend the known features of GA structure which favour flowering relative to stem elongation in L. temulentum. Moreover, C-16, 17-dihydro-GA₅ mimics, in its daylength- and wavelength-dependence and lack of stem elongation, characteristics of the LD stimulus in L. temulentum.     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Paracrine control of tissue regeneration and cell proliferation by Caspase-3

Executioner caspases such as Caspase-3 and Caspase-7 have long been recognised as the key proteases involved in cell demolition during apoptosis. Caspase activation also modulates signal transduction inside cells, through activation or inactivation of kinases, phosphatases and other signalling molecules. Interestingly, a series of recent studies have demonstrated that caspase activation may also influence signal transduction and gene expression changes in neighbouring cells that themselves did not activate caspases. This review describes the physiological relevance of paracrine Caspase-3 signalling for developmental processes, tissue homeostasis and tissue regeneration, and discusses the role of soluble factors and microparticles in mediating these paracrine activities. While non-cell autonomous control of tissue regeneration by Caspase-3 may represent an important process for maintaining tissue homeostasis, it may limit the efficiency of current cancer therapy by promoting cell proliferation in those cancer cells resistant to radio- or chemotherapy. We discuss recent evidence in support of such a role for Caspase-3, and discuss its therapeutic implication.