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1.
We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS
gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria
and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l
sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin.
The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes
were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and
segregation of the transgenes were demonstrated by molecular and genetic analyses in the T0 and T1 generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants
from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the
frequency of transformation was lower and sterility was more frequent. 相似文献
2.
Scutella of immature embryos from two barley cultivars were used for cell culture and transformation. Explants were supplied by continuous growth of donor plants in a 2-week schedule under defined conditions at first plants were grown for 6–7 weeks in a growth chamber, followed by 10–13 weeks in a greenhouse with stringent control of temperature and light round the year. Strong seasonal variation in plant regeneration frequency was observed for both genotypes in non-bombarded (control) as well as bombarded and subsequent selected explants. Scutella from immature embryos of cv. Salome showed increased frequencies of plant regeneration from January to March, reaching highest values in March/April and followed by a continuous and strong decrease from May to December. This tendency was observed in all 3 years studied, although absolute numbers of plant regeneration varied between the years. The same seasonal effect was evident for plant regeneration from immature scutella of cv. Golden Promise. Frequency of embryogenic callus formation was also found to be influenced by season but this effect was not so pronounced as for plant regeneration. 相似文献
3.
4.
To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones
which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated. 相似文献
5.
We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.Communicated by W. Harwood 相似文献
6.
Ana Sofia Duque Susana de Sousa Araújo Matilde Ataíde Cordeiro Dulce Maria Santos Manuel Pedro Fevereiro 《Plant Cell, Tissue and Organ Culture》2007,90(3):325-330
We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP)
and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method
is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported,
with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated
by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing
the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for
early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the
previously selected embryos. 相似文献
7.
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
8.
Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility. 相似文献
9.
Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent
cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with
emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region
of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis
on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm
in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation
period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants
or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An
optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl−1 hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl−1 hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature
cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols
for transformation of soybean should be established under both Agrobacterium and selection conditions. 相似文献
10.
Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize. 相似文献
11.
Songul Gurel Ekrem Gurel Rajvinder Kaur Joshua Wong Ling Meng Han-Qi Tan Peggy G. Lemaux 《Plant cell reports》2009,28(3):429-444
A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for
example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various
temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal
43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at
43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased
frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs
were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2
to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing
calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar
treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable
transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is
several-fold higher than published frequencies. 相似文献
12.
Andrej Kormutak Božena Vookova Peter Manka Jan Salaj Vladimir Camek Dušan Gömöry 《Trees - Structure and Function》2008,22(5):657-662
Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving
ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged
at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared
species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates
of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship
between Pinus sylvestris and Pinus mugo. 相似文献
13.
Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following
co-cultivation, leaf explants were cultured on selective medium containing 10 mg l−1 hygromycin and 500 mg l−1 cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved
after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength
MS basal medium supplemented with 10 mg l−1 hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical
assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%. 相似文献
14.
Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according
to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c)
large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals
until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total,
2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother
cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present
at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental
stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that
embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number
of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing
endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed. 相似文献
15.
Monika Höfer 《Acta Physiologiae Plantarum》2005,27(4):709-716
Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed. 相似文献
16.
17.
Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve
this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration
from rice shoot apical meristem. By use of MS medium containing 4 mg L−1 thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage.
All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil
within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either
250 mg L−1 carbenicillin or 250 mg L−1 cefotaxime to kill the rice shoot apical meristem was 50 mg L−1 and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency
of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient
GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T0 transformant plantlets. 相似文献
18.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
19.
A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) 总被引:3,自引:0,他引:3
Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic
and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue
culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly
used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The
procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel
tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse
facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves
in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus
for the generation of stably transformed maize plants. 相似文献
20.
The effects of genotype, pollen or growth regulator-pretreatment of pistils, developmental stage of the ovule (embryo sac)
and culture media on induction of gynogenesis, and subsequent plantlet regeneration in vitro were assessed in interspecific
Gossypium barbadense × G. hirsutum cotton hybrids. Gynogenesis occurred in all genotypes used when the pistils had been pre-treated with pollen from Hibiscus cannabinus and ovaries were harvested 5 or 10 days after anthesis. The use of culture media, SH and MS, showed no significant differences
in responding ovules, embryogenic ovules or embryo germination frequency. Recovered progeny were characterized cytogenetically
and microscopically to help documenting their reproductive basis. Root tip chromosome counts of 17 plants established from
ovule culture revealed that chromosome numbers ranged from 27 to 44. Although the reproductive mechanisms need to be characterized
more extensively by cytological and molecular means, the observations suggest that gynogenesis in cotton involves some unusual
reproductive events. Aneuploids could be useful for functional genomic characterization of genome shock, deletion mapping,
and germplasm introgression. 相似文献