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1.
Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa… 相似文献
2.
Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L. 总被引:1,自引:0,他引:1
XIANG Fengning XIA Guangmin CHEN Huimin 《中国科学:生命科学英文版》2003,46(3):243-252
Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants. 相似文献
3.
A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA3 Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid 相似文献
4.
Karin Sonntag Brigitte Ruge-Wehling Peter Wehling 《Plant Cell, Tissue and Organ Culture》2009,96(3):297-305
A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all
tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks)
and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown
pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids.
However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast
fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration. 相似文献
5.
In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated
protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat
protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones
were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall
fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with
or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism,
and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained
introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there
were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid
clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration. 相似文献
6.
In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric
fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin
(Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm−2 led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when
they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed
extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between
UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed
to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots
were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism
(AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism
sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp)
and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated
that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel
germplasms, which may provide an alternative approach for cultivar improvement. 相似文献
7.
Silvia Flores-Benítez Juan F. Jiménez-Bremont Sergio Rosales-Mendoza Gerardo R. Argüello-Astorga Rosalba Castillo-Collazo Ángel Gabriel Alpuche-Solís 《Plant Cell, Tissue and Organ Culture》2007,91(3):215-224
Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and
embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants
was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving
a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive
with the GUS assay after 14 months on selective medium while still undergoing regeneration. 相似文献
8.
Summary An efficient system for the regeneration of plants from protoplasts was developed in Alstroemeria. Friable embryogenic callus (FEC) proved to be the best source for protoplast isolation and culture when compared with leaf
tissue and compact embryogenic callus. Protoplast isolation was most efficient when FEC was cultured under vacuum for 5 min
in an enzyme solution consisting of 4% cellulase, 0.5% Driselase and 0.2% Macerozyme, followed by culture for 12–16h in the
dark at 24°C. Cell wall formation and colony formation were better in a liquid medium than on a semi-solid agarose medium.
Micro-calluses were formed after 4 wk of culture. Ninety percent of the micro-calluses developed into FEC after 12wk of culture
on proliferation medium. FEC cultures produced somatic embryos on a regeneration medium and half of these somatic embryos
developed shoots. Protoplast-derived plants showed more somaclonal variation than vegetatively propagated control plants. 相似文献
9.
We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
10.
Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM8P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid,
2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 105 cm−3 was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency
of 9.12 ± 2.61 % at the 40th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were
cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo
induction was further increased by addition of 0.14 μM gibberellic acid. 相似文献
11.
We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- 6-BAP
6-benzylaminopurine
- PP
Protoplasts 相似文献
12.
Y. Y. Wu Q. J. Chen X. H. Cui H. Chen J. Chen X. C. Wang 《Russian Journal of Plant Physiology》2007,54(4):524-529
We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were
tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest
callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine.
Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized
regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin,
a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques,
we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results
indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and
transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596.
The text was submitted by the authors in English. 相似文献
13.
Ananya Paul Kalyan Mitter Sarmistha Sen Raychaudhuri 《Plant Cell, Tissue and Organ Culture》2009,97(3):303-311
The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica
charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction
of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell
division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos
of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli
(5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic
media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented
with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous
free PA levels. 相似文献
14.
Anika Nadja Sabine Prange Margrethe Serek Melanie Bartsch Traud Winkelmann 《Plant Cell, Tissue and Organ Culture》2010,101(2):171-182
Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations
of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic
embryos per gram fresh mass. Up to 4.24 × 105 protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived
from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis.
Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the
protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated
from the embryogenic cultures. 相似文献
15.
Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants. 相似文献
16.
Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in
thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 106/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l
α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis
of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l
zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS)
medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid.
Regenerated plants have normal morphology. 相似文献
17.
Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn. 相似文献
18.
Patrícia Monah Cunha Bartos Hugo Teixeira Gomes Sueli Maria Gomes Sebastião Carvalho Vasconcelos Filho João Batista Teixeira Jonny Everson Scherwinski-Pereira 《Biologia》2018,73(12):1255-1265
The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants. 相似文献
19.
Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O·Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O·Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.Abbreviations BAP 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid 相似文献
20.
Vanessa Endress Jorge Barriuso Pilar Ruperez Juan Pedro Martin Antonio Blazquez Nieves Villalobos Hilario Guerra Luisa Martin 《Plant Cell, Tissue and Organ Culture》2009,97(3):323-329
Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole
explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins,
and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the
highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the
highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to
a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli,
primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides
consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli. 相似文献