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1.
Mei-Lie M. C. Tan Ellen M. Rietveld Gijsbert A. M. van Marrewijk Ad J. Kool 《Plant cell reports》1987,6(3):172-175
Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 l droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).Abbreviations BAP
6-Benzylamino purine
- 2,4-D
2,4-dichlorophenoxy acetic acid
- IAA
indole acetic acid
- MES
2-(N-morpholino)- ethane sulfonic acid
- NAA
naphthalene acetic acid
- PE
plating efficiency 相似文献
2.
Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP
6-benzylaminopurine
- NAA
-naphthaleneacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- MS
Murashige and Skoog (1962)
- KM
Kao and Michayluk (1975)
- g.f.wt.
gram fresh weight 相似文献
3.
High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species 总被引:1,自引:0,他引:1
P. van der Valk O. E. Scholten F. Verstappen R. C. Jansen J. J. M. Dons 《Plant Cell, Tissue and Organ Culture》1992,30(3):181-191
The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- VDH
Van Der Have Seed company 相似文献
4.
Summary Mesophyll protoplasts of an interspecific Lycopersicon esculentum Mill, (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 105/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium.Abbreviations ABA
abscisic acid
- BAP
6 benzylaminopurine
- 2,4-D
2,4 dichlorophenoxy acetic acid
- DMSO
dimethyl sulfoxide
- GA3
gibberellic acid
- GOT
glutamate oxaloacetate
- IAA
indoleacetic acid
- IBA
indolebutyric acid
- IDH
isocitrate dehydrogenase
- MDH
malate dehydrogenase
- MES
morpholinoethane-sulfonic acid
- PEG
polyethylene glycol
- 6-PGDH
6 phosphogluconate dehydrogenase
- PGI
phosphoglucoisomerase 相似文献
5.
Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (>9.0×106 g f.wt.-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.Abbreviations BAP
6-benzylaminopurine
- 2,4-d
2,4-dichlorophenoxyacetic acid
- g f.wt.
gram fresh weight
- IAA
indoleacetic acid
- MS
Murashige & Skoog (1962)
- NAA
-naphthaleneacetic acid
- Zea
zeatin 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):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
7.
Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP
6-benzylaminopurine
- IAA
3-indoleacetic acid
- MS
Murashige and Skoog (1962)
- NAA
1-naphthylacetic acid
- Pfr
Photon fluence rate
- V-KM
Binding and Nehls (1977) 相似文献
8.
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
9.
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
10.
Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP
6-benzylaminopurine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- DPX
dibutylphthalate xylol
- MS
Murashige and Skoog (1962) basal medium
- NAA
1-Naphthaleneacetic acid
- PAA
Phenylacetic acid 相似文献
11.
Helianthus maximiliani is one of the wild Helianthus species with the genes for resistance to many pathogens including Sclerotinia sclerotiorum. Unfortunately, a transfer of disease resistance genes from this species into the cultivated sunflower is limited by its poor crossability with the cultivated sunflower and sterility of interspecific hybrids. To overcome this problem, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clone of H. maximiliani were electrically fused with etiolated hypocotyl protoplasts of the cultivated sunflower inbred line PH-BC1-91A. Fusion products were embedded in agarose droplets and subjected to different regeneration protocols. Developed microcalluses were released from the agarose and transferred into solid media. Shoot regeneration was achieved by culture of calluses on regeneration medium containing 2.2 mg l−1 BAP and 0.01 mg l−1 NAA after the treatment with a high concentration of 2,4 D for a limited period of time. A morphological and RAPD analysis confirmed a hybrid nature of the regenerated plants. 相似文献
12.
An improved system involving a modification of the bead culture system was developed for culturing pea protoplasts. Using this method, sustained divisions and callus growth could be obtained in all 10 cultivars tested. In the best responding cultivar division frequency could be raised from 17% in liquid culture to 80% in the bead system. Shoot regeneration with a reproducible frequency of about 1% could be obtained from protoplast-derived calli in two of the tested cultivars.Abbreviations ABA
abscisic acid
- BA
N6-benzyladenine
- NAA
-naphthylacetic acid
- 2,4-D
2,4-dichlorophenoxy-acetic acid 相似文献
13.
Protoplasts isolated from cotyledon-derived callus of Actinidia chinensisPlanch. var. chinensis (2N=2x=58) were fused with mesophyll protoplasts of Actiniadia kolomikta(Maxim. et Rupr.) Maxim (2N=2x=58) using a PEG method. Plantlets were regenerated from the fusion product clone 11. RAPD analyses, chromosome numbers of root tip cells and fluorescence peak position of leaf nuclei confirmed that clone 11 was an interspecific somatic hybrid (2N=4x=116) between A. chinensis and A. kolomikta. The chilling tolerance of the somatic hybrid was tested with in vitro leaves at low temperatures. Based on data of leaf thickness, electroconductivity, proline levels, malondialdehyde content and activity of superoxide dismutase, dendrogram cluster analysis suggested that the interspecific somatic hybrid was similar to A. kolomikta, and might have a higher capacity of cold resistance than A. chinensis. 相似文献
14.
A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl
to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration
frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium
supplemented with 3 mg dm−3 6-benzylaminopurine (BA) and 0.1 mg dm−3 naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the
number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive
for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response.
The addition of 3.0 mg dm−3 AgNO3 was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium. 相似文献
15.
M. M. C. Tan C. M. Colijn-Hooymans W. H. Lindhout A. J. Kool 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1987,75(1):105-108
Summary Shoot regeneration from leaf discs and leaf mesophyll protoplasts of 11 genotypes of Lycopersicon esculentum (the cultivated tomato), were compared. In both regeneration procedures genotypic differences were observed between inbred lines, and also between F1 hybrids and their parental lines. In the tested hybrid genotypes no heterosis effect with respect to shoot regeneration capacity was observed. A correlation between shoot regeneration from leaf discs and from leaf mesophyll protoplasts was apparent in the tested genotypes. This suggests that using the described procedure, shoot regeneration from leaf discs can be usef for rapid pre-screening for regeneration capacity from protoplasts of tomato genotypes. 相似文献
16.
Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D
2,4-dichlorphenoxyacetic acid
- NAA
naphthalene acetic acid
- IAA
indole-3-acetic acid
- BAP
6-banzylaminopurine
- IPA
isopentenyladenine
- IPAR
isopentenyladenosine
- MES
(2-[N-morpholino]) ethanesulfonic acid 相似文献
17.
Protoplasts were isolated from field and in vitro-grown leaves, cotyledons and cell suspension cultures (of ovule callus origin) of the scion apple cultivars Starkrimson, Rainier, Qiujin and Liaofu. Fast-growing calluses were obtained from leaf, cotyledon and cell suspension derived protoplasts of the four genotypes. The best proliferation responses were obtained from cell suspension protoplasts. For all genotypes tested, nodular calluses were obtained from protoplasts that had originally been cultured on K8P medium, but only those of cultivar Starkrimson underwent organogenesis. In this cultivar shoot buds were produced on callus derived from both cotyledon and cell suspension protoplasts and complete plants. This is the first example of whole plant regeneration from protoplasts isolated from an undifferentiated tissue in apple.Abbreviations BA
6-benzylaminopurine
- 2,4-d
2,4-dichlorophenoxyacetic acid
- IAA
3-indole acetic acid
- IBA
3-indole butyric acid
- LH
lactalbumin hydrolysate
- MS
Murashige & Skoog (1962)
- NAA
1-naphthaleneacetic acid
- TDZ
thidiazuron
- VC
L(+) ascorbic acid 相似文献
18.
F. H. M. Derks A. Zelcer C. M. Colijn-Hooymans 《Plant Cell, Tissue and Organ Culture》1990,21(3):211-216
Mesophyll protoplasts from in vitro grown plants of a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry were isolated with yields between 0.4 to 4.4 × 106 protoplasts per gram leaf tissue. Success in the culture of these protoplasts was dependent on embedding of the protoplasts in 100 µ1 agarose droplets 0.6% (w/v). A plating efficiency of 4.0% was obtained when the protoplasts were cultured in TM-2 medium with sucrose concentrations of 8.7 to 9.6% (w/v) resulting in an osmotic pressure of 432 to 469 mOsmol kg-1. After 14 days of protoplast culture, microcalli with a diameter of 3 mm were observed. After 3 weeks, macrocalli were obtained which were transferred to regeneration medium. Regeneration of shoot primordia, with a frequency of 19%, was obtained on TM-4 medium supplemented with 1% (w/v) sucrose. The first shoot primordia were visible 10 weeks after protoplast plating. For development of the shoot primordia into shoots it was necessary to increase the sucrose concentration to 6% (w/v). Eight out of eleven regenerants were diploid (2n = 2x = 24); the other three were tetraploid. Efficient regeneration of mesophyll albino protoplasts from tomato opens the way to select at the cellular level for the chloroplast transfers. 相似文献
19.
E. A. Matibiri S. H. Mantell 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(8):1017-1022
An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed. 相似文献
20.
M. Nakano M. Mii 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(1):1-5
Summary Protoplasts isolated from leaf mesophyll cells of Dianthus chinensis and D. barbatus were fused by polyethylene glycol (PEG). Calli exhibiting vigorous growth were selected from the PEG-treated protoplasts and shoots were regenerated from one of these calli after 5 months of culture. These shoots readily rooted and continuously produced flowers in the in-vitro condition. The data on flower color, chromosome number, and esterase isozyme patterns indicated that this plantlet was an interspecific somatic hybrid. The hybridity of the plantlet was also confirmed by nuclear rDNA analysis. This report provides the possibility of applying the somatic hybridization technique for the genetic improvement of the genus Dianthus. 相似文献