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1.
Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP
N6-benzylaminopurine
- BM
basal medium
- 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
1-naphthalene acetic acid
- PEG
polyethylene glycol
- PKM
modified Kao (1977) medium for protoplast culture 相似文献
2.
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 相似文献
3.
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) 相似文献
4.
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 相似文献
5.
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 RO plants was about 40%. 相似文献
6.
Callus production from willow (Salix viminalis L.) protoplasts 总被引:2,自引:0,他引:2
Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 106 ± 1.9 · 106 protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 106 protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA
benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- MS medium
Murashige & Skoog (1962) medium
- WP medium
Woody Plant medium (Lloyd & McCown 1981) 相似文献
7.
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 相似文献
8.
Tang K. Sun X. An D. Power J.B. Cocking E.C. Davey M.R. 《Plant Cell, Tissue and Organ Culture》2000,60(1):79-82
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. 相似文献
9.
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. 相似文献
10.
Daina H. Simmonds Nancy E. Long Wilfred A. Keller 《Plant Cell, Tissue and Organ Culture》1991,27(3):231-241
Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA
N6-benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- NAA
naphthaleneacetic acid 相似文献
11.
Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×106/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R1 plants were self-pollinated and immature R2 embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R2-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC
backcross
- BMS
Black Mexican Sweetcorn
- 2,4-D
2,4-dichlorophenoxyacetic acid
- PWS
protoplast wash solution (0.2 M mannitol, 80 mM CaCl2)
- FDA
fluorescein diacetate
- ABA
abscisic acid 相似文献
12.
Protoplasts isolated from mesophyll cells of Eruca sativa Lam., cultured on suitable medium, underwent sustained cell divisions to form calli. The plating efficiency was found to be 0.4%. The protoplast-derived calli subsequently produced plantlets through organogenesis (15.71%) and somatic embryogenesis (11.25%). Regenerated plants exhibited normal appearance. These results indicate potential to introgress desirable traits from this wild crucifer into important oilseed and cole Brassicas by protoplast fusion and hybrid recovery.Abbreviations B5
Gamborg et al., 1968
- K3
Kao and Michayluk, 1974
- MS
Murashige and Skoog, 1962
- BAP
6-benzylaminopurine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
1-naphthalene acetic acid
- GA3
Gibberellic acid 相似文献
13.
E. G. M. Meijer F. van Iren E. Schrijnemakers L. A. M. Hensgens M. van Zijderveld R. A. Schilperoort 《Plant cell reports》1991,10(4):171-174
A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15–21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.Abbreviations DMSO
dimethyl sulfoxide
- 2,4-D
2,4-dichlorophenoxyacetic acid
- NAA
1-naphtylacetic acid
- FDA
fluorescein diacetate 相似文献
14.
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 相似文献
15.
A. C. Bonfils S. C. Gleddie J. A. Webb W. A. Keller 《In vitro cellular & developmental biology. Plant》1992,28(3):137-142
Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but
underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in
liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium.
These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants
were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast
viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated
on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions
were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were
obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos
previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s
purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization. 相似文献
16.
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. 相似文献
17.
H. Ichikawa L. Tanno-Suenaga J. Imamura 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1987,74(6):746-752
Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion. 相似文献
18.
Protoplasts were isolated from leaf mesophyll and cell suspensions of two accessions of Stylosanthes guianensis (Aubl.), a tropical forage legume. When cultured in VKM liquid culture medium, both types of protoplasts divided at a rate of 4–8%, and subsequently formed cell colonies. Protoplast-derived calluses produced numerous shoots when transferred to regeneration medium. Regenerated shoots could be easily rooted, and plantlets were transferred to soil. The effects of several factors on the efficiency of this protoplast system have been investigated.Abbreviations BAP
6-benzylaminopurine
- GA3
Gibberellic acid
- NAA
Naphthalene acetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- Zea
Zeatin 相似文献
19.
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 相似文献
20.
Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p
culture media; see Material and methods
- cv
chltivar
- dicamba
3,6-dichloro-o-anisic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- picloram
4-amino-3,5,6-trichloropicolinic acid 相似文献