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1.
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
2.
An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts 总被引:2,自引:0,他引:2
An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform
protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation
in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×104 protoplasts ml−1 of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid
medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording
the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts
in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore,
this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
3.
A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA. 相似文献
4.
J. R. Myers J. W. Grosser N. L. Taylor G. B. Collins 《Plant Cell, Tissue and Organ Culture》1989,19(2):113-127
Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613 相似文献
5.
Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated
calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as
well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration
medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared
to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the
regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three
months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts
as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
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
7.
E B. d'Utra Vaz I. H. Slamet A. Khatun E. C. Cocking J. B. Power 《Plant cell reports》1992,11(8):416-418
The effect of a molecular oxygen atmosphere on protoplast culture, compared with normal cultural procedures, was investigated for tomato, rice and jute protoplast systems. Protoplasts, in unsealed Petri dishes were cultured in a sealed 250 ml volume container in which the atmosphere was replaced with sterile molecular oxygen (O2 100% v/v) achieved by a one minute oxygen flow (10 mBar). Controls were cultured in a similar way but with a normal atmosphere (20.5% v/v oxygen). For all three species a major improvement in plating efficiency and colony formation was observed in the oxygen-enriched atmospheres. In the case of rice, cultivar Taipei 309, a significant increase in plant regeneration capacity, from protoplast-derived colonies, was observed for the high oxygen cultures.Abbreviations MES
2-N-morpholinoethane sulfonic acid
- PE
plating efficiency 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.
Traud Winkelmann Janine Specht Margrethe Serek 《Plant Cell, Tissue and Organ Culture》2006,86(3):337-347
A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 105 protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.8 mg l−1 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil. 相似文献
10.
János Pauk Zoltán Kertész Barnabás Jenes László Purnhauser Outi Manninen Seppo Pulli Zoltán Barabás Dénes Dudits 《Plant Cell, Tissue and Organ Culture》1994,38(1):1-10
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. 相似文献
11.
Bolandi A.R. Branchard M. Alibert G. Serieys H. Sarrafi A. 《Plant Cell, Tissue and Organ Culture》1999,57(3):189-193
Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with
a density of 2×104 protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets
of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media
were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced
were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from
0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype
for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly
higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among
genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions.
Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
12.
Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old
callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting
of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l-1 kinetin. The planting density was 2.5–3.0×105 protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating
efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content.
A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485).
Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content.
Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success
in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
13.
Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
6-benzylaminopurine
- NAA
naphthaleneacetic acid
- L1, L2
medium according to Lazzeri et al. 1991
- L3 medium
medium according to Jähne et al. 1991a 相似文献
14.
Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW
fresh weight
- MS
Murashige and Skoog (1962)
- 1/2MS medium
MS medium containing half strength mineral salts
- 1/2MS-0
1/2MS medium containing no growth regulators
- NAA
1-naphthaleneacetic acid
- p-calli
protoplast-derived calli
- PE
plating efficiency
- picloram
4-amino-3,5,6-trichloro-picolinic acid 相似文献
15.
Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum 总被引:3,自引:0,他引:3
Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 103 per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H2 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 104 cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration. 相似文献
16.
Protoplasts were isolated from leaves of in vitro-grown shoot cultures of different Petunia and Calibrachoa genotypes by enzyme digestion with 0.6% macerozyme R-10 and 2.0% cellulase R-10. Shoot regeneration was achieved in five
out of nine Calibrachoa and three out of four Petunia genotypes. Protoplast yield and frequency of shoot regeneration varied among experiments and genotypes. Among all regenerants,
few morphological changes, such as chlorophyll deficiency, variegated leaves, and polyploidization, were observed. 相似文献
17.
Through induction of totipotent stage in cultured cells plants were regenerated from protoplast derived callus colonies of potato. Growing of the plants in vitro with optimal aeration and pretreatment of the leaves in dark and cold prior to protoplast isolation has improved the protoplast yield and frequency of cell division. Protoplasts of four potato genotypes have started to divide within 2-5 days after plating them into culture medium containing 2,4-D (0.2 mg/l); ZEA (0.5 mg/l); and NAA (1 mg/l) as growth regulators. Embedding of the cells into agarose proved to be favourable to avoid cell browning and to increase colony formation. The series of hormone treatments based on complex action of NAA and BAP promoting colony growth and greening, ZEA and IAA inducing shoot redifferentiation, and GA3 plus NAA supporting shoot elongation and rooting, finally resulted in high frequency of plant regeneration from microcolonies. 相似文献
18.
Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For
each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant
regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical
factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain
seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the
sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration
in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the
gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization. 相似文献
19.
High frequency plant regeneration from rice protoplasts by novel nurse culture methods 总被引:5,自引:0,他引:5
Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes. 相似文献
20.
Protoplasts of Vitis rotundifolia Michx. cv. Summit were isolated from mesophyll of axenic shoot cultures under different enzyme concentrations and digestion times. Viability and plating efficiency were assessed and related to the cortical microtubule network, visualized using immunofluorescence. Higher concentrations of enzyme isolation medium significantly decreased protoplast viability and plating efficiency. However, the cortical microtubule network appeared stable, at all concentrations with dense, continuous microtubule strands in both random and parallel arrays. In contrast, longer vs shorter enzyme incubation duration resulted in significantly lower plating efficiency, which was correlated with changes in cortical microtubule organization. With longer incubation, the frequency of parallel microtubule strands decreased; microtubule organization showed increasing disruption, microtubule strands were shortened, fragmented and exhibited only a weak fluorescence labeling. Both high enzyme concentration and prolonged incubation periods negatively affected protoplast regenerability, but in different ways. Microtubule organization was sensitive to duration of incubation, but not to enzyme concentration. It is concluded that the presence of a well-developed cortical microtubule network does not gurantee regeneration. Other factors related to isolation appear to be involved. 相似文献