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1.
Paul W. J. Taylor Hian-Lien Ko Stephen W. Adkins Carl Rathus Robert G. Birch 《Plant Cell, Tissue and Organ Culture》1992,28(1):69-78
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. 相似文献
2.
Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures. 相似文献
3.
Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS
Murashige and Skoog (1962)
- FW
fresh weight
- MES
2-N-morpholinoethane sulfonic acid
- FDA
fluoroscein diacetate
- NAA
1-naphthaleneacetic acid 相似文献
4.
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
5.
Kexuan Tang Quanan Hu Enpeng Zhao Aizhong Wu 《In vitro cellular & developmental biology. Plant》2000,36(4):255-259
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. 相似文献
6.
Kong-Nan Zhao Dennis J. Bittisnich Gerald M. Halloran Malcolm I. Whitecross 《Plant Cell, Tissue and Organ Culture》1995,40(1):59-72
Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA
benzylaminopurine
- CPW
Composition of Protoplast Washing-solution
- CW
Calcolfluor White
- EDTA
ethylenediamine-tetraacetic acid
- KT
Kinetin
- Md MS
modified Murashige and Skoog medium
- 2,4-d
2,4-dichlorophenoxyacetic acid
- NAA
-naphthaleneacetic acid
- IAA
indole-3-acetic acid
- PAR
photosynthetically active radiation
- SDS
sodium dodecyl sulfate 相似文献
7.
Both the stage of the growth cycle and the age of the cell culture used to isolate protoplasts had a pronounced effect on both transient and stable expression of the GUS gene. A level of GUS gene transient expression of 9000 pmol 4MU/μg protein/h and a frequency of GUS gene stable expression of 5.72% were obtained with protoplasts isolated from suspension cultures 10–20 weeks after initiation and 3–4 days after subculturing when an optimized transformation protocol and a rice actin 1 promoter-uidA gene construct were used. The effect of the cell growth cycle on GUS gene transient expression was closely correlated with the growth rate and the rate of protein synthesis in cell cultures whereas prolonged subculturing of the cells resulted in a gradual decline in both transient and stable expression. The length of time cells were digested in cell wall digestion enzyme and the osmolarity of the transformation medium were found to critically affect both the level of transient and stable GUS gene expression. The composition and osmolarity of the protoplast culture medium was less critical for transient GUS gene expression although the osmolarity of the medium was shown to have a significant effect on stable expression of the GUS gene. 相似文献
8.
Li Hongchao Machii Hiroaki Hagio Takashi Takezaki Akane Hirabayashi Toshio 《Plant Cell, Tissue and Organ Culture》1999,58(2):119-125
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. 相似文献
9.
Kong-Nan Zhao Dennis J. Bittisnich Gerald M. Halloran Malcolm I. Whitecross 《Plant Cell, Tissue and Organ Culture》1995,40(1):73-84
Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d
2,4-dichlorophenoxyacetic acid
- BA
benzylaminopurine
- EDTA
ethylenediaminetetraacetic acid
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- KT
kinetin
- GA3
gibberellic acid
- MS
Murashige and Skoog medium
- NAA
-naphthaleneacetic acid
- PAR
photosynthetically active radiation 相似文献
10.
Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts 总被引:5,自引:2,他引:3
A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3′ chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system. 相似文献
11.
Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 106 protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research. 相似文献
12.
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
13.
《Plant science》1986,46(2):133-142
Leaf protoplasts of Populus alba L. × P. grandidentata Michx. (NC-5339) were isolted from shoot cultures of non-seedling origin and cultured through plant regeneration. Complete protoplast development was dependent on providing a stress-free culture environment which included eliminating ammonium, agar, exudate build-up, and light during the culture period. Contact with a solid surface appeared to stimulate development and thus the protoplasts were cultured in a liquid floating-disc system in which they adhered to the fibers of a polyester screen. Protoplasts exhibited a slow, staged development which resulted in cell division 6 weeks following protoplast isolation. The resulting colonies proliferated rapidly and rooted spontaneously. Shoot regeneration occurred when the protoplast-derived calli were exposed to thidiazuron, and such shoots could be readily rooted. This is the first report of reproducible plant regeneration from leaf protoplasts of non-seedling origin of a tree species. 相似文献
14.
Plant regeneration from highly embryogenic callus,cell suspension and protoplast cultures of Trifolium fragiferum 总被引:1,自引:0,他引:1
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. 相似文献
15.
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 相似文献
16.
将谷子胚性愈伤组织粘液提取物添加到谷子原生质体培养基中,其对原生质体培养的影响表明该提取物有助于原生质体形成细胞壁;并且该类有粘液分泌的念伤组织的原生质体游离所需的酶液浓度低、处理时间短。由原生质体形成完整细胞的数量在一定范围内与谷子原生质体培养的植板率相对应;通过增加形成完整细胞的数量可较大幅度地提高原生质体培养的植板率。 相似文献
17.
R Butowt A Niklas MI Rodriguez-Garcia A Majewska-Sawka 《Journal of plant research》1999,112(1):107-116
Beta vulgaris L.). The spatial and temporal expression of both antigens was studied in suspension cells used as the source-tissue for protoplast
isolation, in suspension- and mesophyll-derived protoplasts, and in cells which developed from both types of protoplast. Immunofluorescence
and immunocytochemical-electron microscopic methods revealed that labeling was present in the cell walls of most suspension
cells and also in the incipients of cell walls synthesized around the protoplasts. This signal became much more intense as
rebuilding of the cell wall progressed during culture. Relatively weaker labeling was observed in the cytoplasm, where it
was frequently associated with the vacuolar compartment. Signal intensity varied between individual cells of the same population
and in successive stages of development, but was always stronger with JIM13 than with JIM8. The role of JIM13-responsive epitope
in the development of suspension-derived protoplasts was further studied by its ability to bind antibody added to cultures
of different ages. Both JIM8- and JIM13-responsive epitopes were widespread in sugar beet cells of different origin and stage
of cell wall synthesis. These epitopes may play an important role in cell wall formation and growth under in vitro conditions.
Received 17 August 1998/ Accepted in revised form 13 January 1999 相似文献
18.
Summary Mesophyll protoplasts were isolated from axenic shoot cultures of pear cultivars, exhibiting different degrees of susceptibility to fire blight infection at the whole plant level and they were co-cultured with the wild-type strain CFBP 1430 of Erwinia amylovora, and with an avirulent transposon mutant of the former (PMV 6046). Results, as assessed in terms of the effects of bacteria on protoplast viability, the time to the onset of divisions, the percentage of the originally cultivated protoplasts that divided once and of those proliferating to give 10-cell colonies, correlated with field resistance to fire blight of the respective pear genotypes. These results might provide a model for a better understanding of the interaction between pear and E. amylovora.Abbreviations BAP
6-benzylaminopurine
- FDA
fluorescein diacetate
- fwt
fresh weight
- IAA
indole-3-acetic acid
- IPE
initial plating efficiency
- MPE
intermediate plating efficiency
- MS
Murashige and Skoog (1962)
- NAA
1-naphthaleneacetic acid
- PVP-10
polyvinylpirrolidone (av.molecular weight 10000)
- uv
ultra-violet 相似文献
19.
Craig R. Landgren 《American journal of botany》1976,63(4):473-480
Mitotic figures of diploid, tetraploid, octaploid and 16-ploid nuclei were observed in cultures of pea root protoplasts whose initial DNA content was apparently 2C and 4C. The distribution of these mitotic figures in the different ploidy levels paralleled the distribution of mitotic figures in the culture of intact root explants and may be related to the hormonal stimulation of mitoses in these cultures. The patterns of the time course of both DNA synthesis and cell division in the protoplast cultures were similar to such patterns observed in the culture of intact root explants, although longer lag periods were observed in the protoplast cultures. Mitotic abnormalities including both chromosome breakage and spindle disfunction were observed in protoplast cultures. A large portion of the cell pairs derived from mitoses (27 % in one experiment) contained Feulgen-positive micronuclei. An accumulation of an as yet unidentified differentiation product termed dense cytoplasmic protoplast derivative was observed. Some of the conditions influencing the development of these derivatives are reported. 相似文献
20.
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