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1.
G. Manders W. C. Otoni F. B. d'Utra Vaz N. W. Blackball J. B. Power M. R. Davey 《Plant cell reports》1994,13(12):697-702
Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 M 6-benzyl-aminopurine and supplemented with 86 M kanamycin sulphate. The four plants were rooted by transfer to MS based medium with 14.7 M 3-indolebutyric acid and 2.68 M -naphthyleneacetic acid for 7 d, followed by MS based medium lacking growth regulators. Both media used for rooting contained 172 M kanamycin sulphate. Rooted plants were potted and grown to maturity. Three of the plants synthesised nopaline and expressed neomycin phosphotransferase activity; DNA dot blot and polymerase chain reaction analyses confirmed the presence of the neomycin phosphotransferase gene in three plants.Abbreviations BAP
6-benzylaminopurine
- CTAB
hexadecy-Itrimethylammonium bromide
- EDTA
ethylenediaminetetraacetic acid
- IBA
3-indolebutyric acid
- MS
Murashige and Skoog (1962)
- NAA
-naphthyleneacetic acid
- NPTII
neomycin phosphotransferase II
-
nptII
neomycin phosphotransferase II gene
- PCR
polymerase chain reaction
- SDS
sodium dodecyl sulphate
- Tris
tris(hydroxymethyl)aminomethane 相似文献
2.
High yields of protoplasts were obtained from mesophyll tissue of Digitalis obscura L. Osmotic potential of the isolation medium and Ca2+ were important in obtaining a high viability of the preparations. In different culture techniques employed, liquid-over-agar-solidified medium was superior to liquid medium alone. Agar plating technique was ineffective. On Murashige and Skoog modified medium with casein hydrolysate and several indoleacetic acid and benzyladenine combinations, isolated protoplasts underwent sustained mitotic division and produced calli. The calli formed shoots when transferred to regeneration media. Regenerated shoots could be easily rooted and developed into whole plants on half-strength Murashige and Skoog hormone-free medium. 相似文献
3.
Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium 总被引:1,自引:0,他引:1
Hisato Kunitake Toshiki Nakashima Kinya Mori Masanobu Tanaka Masahiro Mii 《Plant Cell, Tissue and Organ Culture》1995,43(1):59-65
Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3–4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50–100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l–1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control.Abbreviations BA
6-benzylaminopurine
- NAA
1-naphthaleneacetic acid
- MS
Murashige & Skoog (1962) medium
- IBA
indolebutyric acid
- MES
2-N-morpholinoethane sulfonic acid 相似文献
4.
M. A. K. Azad S. Yokota F. Ishiguri N. Yoshizawa 《In vitro cellular & developmental biology. Plant》2006,42(6):502-507
Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast
isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum.
The highest viability (86%) with a yield of 7.1×105 protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained
cell division and colony formation from the protoplasts were best supported at a plating density of 4×105−6×105 protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing
0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived
colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions. 相似文献
5.
A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated. 相似文献
6.
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
7.
Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×105 protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS2 medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP
6-benzylaminopurine
- GA3
Gibberellic acid
- A
Kao and Michayluk, 1981
- KM
Kao and Michayluk, 1975
- MK3
Modified K3
- M8P
Modified 8P
- MS
Murashige and Skoog, 1962
- NAA
1-naphthalene acetic acid
- PE
Plating efficiency 相似文献
8.
《Plant science》1987,53(3):257-262
Conditions were developed for the isolation, culture and regeneration of mesophyll protoplasts of the tree legume, Pithecellobium dulce Benth. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) was essential to induce initial cell divisions and addition of naphthaleneacetic acid (NAA) improved the response. Sustained division and cell colony formation were achieved from the protoplasts cultured in a modified KM8P medium containing 2,4-D (2.3 μM), NAA (3 μM) and benzyladenine (BA) (2.3 μM). Dilution of the osmotica included in the protoplast culture medium was necessary to induce sustained proliferation of the protoplast-derived cells. Differentiation of shoots from the protoplast-derived calli occurred on Murashige and Skoog (MS) medium supplemented with BA (5 μM) and indole-3-acetic acid (1 μM). Omission of 2,4-D from the culture medium, after the initial 2 weeks of protoplast culture, was obligatory to induce shoot morphogenesis. 相似文献
9.
Large populations of viable protoplasts were released from suspension cultured cells of the woody medicinal plant Solanum dulcamara (bittersweet, woody nightshade) when the cells were harvested 3 to 7 months after culture initiation and 4 to 5 days after transfer to fresh medium. A Bio-Gel p6 purified enzyme mixture enhanced the protoplast plating efficiency 6 fold compared to the unpurified mixture, without affecting protoplast yield. Agarose-solidified medium markedly improved protoplast division and colony formation, and enabled protoplasts to be plated at lower densities than in liquid medium. All protoplast-derived tissues produced shoots on MS based medium with 1.0 mgl-1 zeatin. Shoots rooted readily on medium lacking phytohormones. Cytological examination revealed high chromosome stability of suspension cultured cells, of plants derived from such cells, and of protoplast-derived plants. The implication of these results is discussed in relation to the genetic manipulation of this pharmaceutically important plant. 相似文献
10.
Optimal protoplast yields from cotyledons (2.0×106 protoplasts/ 0.5 g tissue) and from true leaves (5.0×106 protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 104 protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA
benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- MS
Murashige and Skoog medium
- NAA
naphthaleneacetic acid 相似文献
11.
Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1–1 4-indole-3yl-butyric acid, 2.0 mg 1–1 BAP, 0.2 mg 1–1 gibberellic acid, 50 mg 1–1 casein hydrolysate and 10 mg 1–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP
6-benzylaminopurine
- FPE
final plating efficiency
- GA3
gibberellic acid
- IAA
4-indole-3yl-acetic acid
- IBA
4-indole-3yl-but yric acid
- IPE
initial plating efficiency
- NAA
1-naphthaleneacetic acid
- f.wt.
fresh weight
- MES
2-N-morpholinoethane sulfonic acid
- MS
Murashige and Skoog (1962)
- %PE
% plating efficiency
- PVP-10
polyvinylpyrrolidone (Av. MW 10,000)
- FDA
fluorescein diacetate 相似文献
12.
The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli. 相似文献
13.
Summary Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing
2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be
transferred to soil after being acclimatized. 相似文献
14.
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
15.
A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture
in B5 medium containing 3 mg l–1
α-naphthaleneacetic acid (NAA) and 1 mg l-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4)
plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l–1 NAA and 1 mg l–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency
of the procedure for protoplast to cell line formation is high (about 80%).
Received: 17 February 1997 / Revision received: 6 November 1997 / Accepted: 15 November 1997 相似文献
16.
《Plant Science Letters》1978,11(2):169-176
A predominantly haploid, approx. 57%, suspension culture of Datura innoxia was obtained and conditions for the isolation, culture and regeneration to shoots of protoplasts from these cells are described.Chromosome counts of protoplast-derived cultures showed that no haploids were present among 15 regenerated shoots, and only 3 of 30 calli contained any haploid tissue. Approx. 9.5% of mitoses examined in a protoplast-derived suspension culture were haploid.The presence of haploid sets of diplochromosomes in the early divisions of protoplasts suggests that endomitosis may be one cause for the observed ploidy increase. 相似文献
17.
Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA
6-benzylaminopurine
- NAA
-naphthaleneacetic acid
- KIN
kinetin
- ZEA
zeatin
- CPW
cell and protoplast wash solution
- B5
Gamborg et al. (1968) mineral elements
- MS
Murashige and Skoog (1962) mineral elements 相似文献
18.
Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA
indol-3-acetic acid
- NAA
naphthalene acetic acid
- 2,4-D
2,4-dichlorophenoxy acetic acid
- KT
kinetin
- BA
6-benzyladenine
- ZT
zeatin
- CH
casein hydrolysate 相似文献
19.
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
20.
A. C. B. de A. Monteiro E. N. Higashi A. N. Gonçalves A. P. M. Rodriguez 《In vitro cellular & developmental biology. Plant》2000,36(6):527-531
Summary Mineral deficiency symptoms were observed in leaves of yellow passionfruit plantlets grown in MS medium (Murashige and Skoog,
1962) with 1.0 mg l−1 (3.0 μM) gibberellic acid. Initially, leaves showed interveinal chlorosis, followed by bleaching of the leaves and retarded growth.
Leaf mineral analysis was done and compared to mineral requirements suggested for passionfruit in the literature. Several
modifications were made to the inorganic composition of MS medium, according to mineral deficiencies, mainly of Fe and Ca,
and possible toxicity of Cl. The concentration of the elements in the new medium (MSM) was based on the mineral composition
of leaves of healthy plants. The chemical equilibrium was checked using the software Geochem (Sposito and Mattigod, 1980)
and final adjustments were made to ensure good availability of nutrients. To test the efficiency of the modified medium nodal
segments were cultured in both MS and MSM supplemented with 3.0 mg l−1 (13.3 μM) 6-benzyladenine. After three subcultures mineral analysis of the leaves was done. Severe mineral deficiency was observed
on the leaves of plantlets cultured in MS, while plantlets cultivated in MSM had green leaves. A comparison of the mineral
analysis of plantlets in both media showed a fairly large increase in Ca, Cu, Fe, Mg and S and decrease in levels of B and
Cl in plantlets cultivated in MSM. A slight increase or decrease in other elements was also observed. Subculture of the chlorotic
plantlets into MSM showed that the visual symptoms of mineral deficiency disappeared in 2–4 wk. 相似文献