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1.
A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment
of embryogenic tissues ( Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines
carrying the hygromycin phosphotransferase ( hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates,
when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure,
transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0
plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model
for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies.
Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998 相似文献
2.
This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination. Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro. Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed. 相似文献
3.
In vitro cultures from two strains of Narcissus confusus (Amaryllidaceae) initiated from mature seeds were screened for their ability to produce alkaloids. Protocols for callus induction,
somatic embryogenesis and organogenesis were established. The alkaloid contents were determined by HPLC. Undifferentiated
calli produced small amounts of galanthamine, which increased with the degree of tissue differentiation. Scanning electron
micrographs of the cultures at different stages of development were made. Embryogenic friable calli were maintained in culture
for more than 2 years, retaining a high regeneration capability. All regenerated plants showed normal morphological characteristics.
Received: 20 August 1997 / Revision received: 20 December 1997 / Accepted: 1 February 1998 相似文献
4.
This review focuses on the improvement of wheat ( Triticum aestivum L.) via tissue and cell culture and its use in gene transfer techniques. Success of the latter critically depends on the ability to regenerate plants from cells or tissues cultured in vitro. Hence, we have devoted attention to the attempts made so far in obtaining regenerants from diverse explants. Although it is known that immature embryos are the best source for initiating morphogenic cultures, basic information related to the process of differentiation can also be gained by studying less responding tissues. The opportunity provided by anther and microspore culture in wheat improvement and the progress made is also presented. To enhance tissue culture responses, identification of chromosomes, gene loci, and genes is of cardinal importance. We have also surveyed the progress made in this regard by conventional but incisive plant-breeding techniques. Gene rearrangements in tissue culture leading to the appearance of somaclonal or gametoclonal variation are of interest in selection of useful cell lines. The last part of the review is devoted to work done on transient gene expression and transformation with emphasis on recent developments. 相似文献
5.
Four biennial species ( Arctium tomentosum, Cirsium vulgare, Dipsacus sylvester and Daucus carota) which originate from habitats of different nutrient availability were investigated in a 2-year experiment in a twofactorial structured block design varying light (natural daylight versus shading) and fertilizer addition. The experiment was designed to study storage as reserve formation (competing with growth) or as accumulation (see Chapin et al. 1990). We show that (i) the previous definitions of storage excluded an important process, namely the formation of storage tissue. Depending on species, storage tissue and the filling process can be either a process of reserve formation, or a process of accumulation. (ii) In species representing low-resource habitats, the formation of a storage structure competes with other growth processes. Growth of storage tissue and filling with storage products is an accumulation process only in the high-resource plant Arctium tomentosum. We interpret the structural growth of low-resource plants in terms of the evolutionary history of these species, which have closely related woody species in the Mediterranean area. (iii) The use of storage products for early leaf growth determines the biomass development in the second season and the competitive ability of this species during growth with perennial species. (iv) The high-resource plant Arctium has higher biomass development under all conditions, i.e. plants of low-resource habitats are not superior under low-resource conditions. The main difference between high- and low-resource plants is that low-resource plants initiate flowering at a lower total plant internal pool size of available resources. 相似文献
6.
A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG)
expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter
and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed
that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining
of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature
of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was
generally present at a higher copy number than the selected (HYG) gene.
Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998 相似文献
7.
In order to take advantage of the high thermostability of its product, β-1,3;1,4-glucanase (lichenase), we used a modified
version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene ( m- licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the
Agrobacterium octopine T R-DNA 2′ gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression
of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various
gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even
after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels
in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.
Received: 4 March 1997 / Accepted: 8 October 1997 相似文献
8.
The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [ Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck× Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration
of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital
marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed
from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus,
most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium
is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy
discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating
the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots
have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors
and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which
is currently a topic of much discussion for the commercialization of transgenic plants.
Received: 28 October 1998 / Accepted: 28 November 1998 相似文献
9.
We tested whether grazing by the specialist beetle Galerucella nymphaeae (Coleoptera: Chrysomelidae) induced resistance to herbivory in the water lily Nuphar luteum macrophyllum (Nymphaeaceae) using both the specialist beetle and the generalist crayfish Procambarus clarkii (Decapoda: Cambaridae). For 2 months, we allowed natural densities of beetles to develop on control plants of Nuphar, while removing beetles every 2–3 days from adjacent plants that were paired by location within our field site. By the end
of the 2-month manipulation, beetle grazing had damaged twice as much leaf surface on control plants as on removal plants
(30.6% vs. 14.2%, respectively). We then offered tissues from control and removal plants to adult and larval beetles and to
crayfish in laboratory assays. Increased levels of previous attack by the specialist beetle either did not affect or increased
water lily attractiveness to beetles, but significantly decreased attractiveness to the generalist crayfish. Beetle larvae
did not feed preferentially on control vs. removal Nuphar in assays using either immature, undamaged leaves that had not yet reached the pond surface or intermediate aged leaves that
had reached the surface and experienced some beetle grazing. Adult beetles consumed significantly more immature leaf tissue
from the heavily grazed controls than from the less grazed removal plants but did not discriminate between control and removal
leaves of intermediate age in either feeding or oviposition preference. In contrast, generalist crayfish consumed significantly
more plant tissue from the less grazed treatment than from the more heavily grazed controls. Crude chemical extracts from
Nuphar strongly deterred crayfish feeding, but neither phenolic content, protein content, nor differential effects of crude extracts
from control vs. removal plants explained crayfish feeding on control versus removal leaves. Our assays suggest that induced
resistance to crayfish may be chemically mediated, but the particular mechanisms producing this response remain unclear. Responses
may be due to defensive metabolites that degrade rapidly following extraction.
Received: 23 July 1997 / Accepted: 8 February 1998 相似文献
10.
Genomic imprinting, where only one of the two parental genes is expressed, occurs in many phyla. In mammals, however, this phenomenon has been primarily studied in mice, and to a lesser extent, in humans. To understand how genomic imprinting may affect development in other species, particularly those with a different mode of placental development from mice and humans, 339 sheep zygotes were micromanipulated to contain either 2 large (presumptive male) or 2 small (presumptive female) pronuclei. One hundred and twenty-seven of these embryos and 86 manipulated and nonmanipulated control embryos were transferred to recipient ewes over 3 breeding seasons. Twenty-one control and 7 experimental conceptuses were recovered on day 21. Four of these conceptuses derived from zygotes with 2 small pronuclei were identified by karyotyping to be gynogenones (maternal-derived genome). While the gross morphology of the embryos appeared no different to those of normal controls, the extra-embryonic tissue from the conceptuses showed some hypertrophy and hypervascularization. Preliminary Northern blots of mRNA from allantoic and trophoblast tissue showed an overexpression of H19 and an underexpression of IGF2. Although the sheep gynogenetic phenotype contrasts with that seen in mice, these two genes appear to be similarly differentially expressed. Mol. Reprod. Dev. 50:154–162, 1998. © 1998 Wiley-Liss, Inc. 相似文献
11.
Transformed rice plants of var `TN1' were regenerated from immature embryos following particle bombardment with a construct
containing the firefly luciferase gene as a reporter gene and the hygromycin resistance gene as a selectable marker. Expression
of the luciferase gene in the presence of the substrate luciferin was visualised in the calli derived from bombarded immature
embryos and in the leaves and roots of the regenerated transformed plants using a low light imaging system (luminograph).
Embryogenic callus proliferation and plant regeneration were unaffected by luciferin treatment and luminograph screening.
The quantitative Luc assay using samples of leaf tissue from the segregating generations gave early information about the
homozygous and hemizygous state of the luc transgene.
Received: 25 August 1998 / Revision received: 2 November 1998 / Accepted: 13 November 1998 相似文献
12.
A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously
difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded
and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII
genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration.
The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot
regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant
PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants
was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were
established in soil and acclimatized in the greenhouse.
Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998 相似文献
13.
Using an improved method for extraction of dsRNA from strawberry leaf tissue, small quantities of several dsRNA species with mol. wt greater than 1.0 × 10 6were detected in strawberry plants free from known strawberry viruses but affected by June yellows (JY). No such dsRNA species were detected in plants of Fragaria vesca or seven strawberry cultivars known to be free from JY. Neither JY symptoms nor these dsRNA species were detected in healthy strawberry and F. vesca plants graft-inoculated with tissue from JY-affected plants. It is not known whether the JY-associated dsRNA species are those of a causal agent of JY or are a consequence of the JY condition. Nevertheless, the detection of such dsRNA species in plants affected by JY may offer a possible objective method for detecting the incipient condition in symptomless strawberry plants. However, the concentrations of dsRNA in JY-affected plants are very low and dsRNA analysis is thought not to be sufficiently reliable for routine testing of plants. The occurrence of anomalous dsRNA species in extracts from some strawberry plants was caused by dsRNA from two-spotted spider mites ( Tetranychus urticae) infesting the plants. 相似文献
14.
Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by
the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed
in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence
made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed
detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic
plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification,
i.e., to alter the emission spectra, would increase its utility.
Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998 相似文献
15.
Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured
woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be
rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability
of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of
pecan [ Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences
between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with
similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated
by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels
of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line
differences and their contribution to phenotypic variation is yet to be determined.
Received: 9 September 1998 / Revision received: 12 November 1998 / Accepted: 8 December 1998 相似文献
16.
The influence of an arbuscular mycorrhizal (AM) fungus on phosphorus (P) and iron (Fe) uptake of peanut ( Arachis hypogea L.) and sorghum ( Sorghum bicolor L.) plants was studied in a pot experiment under controlled environmental conditions. The plants were grown for 10 weeks
in pots containing sterilised calcareous soil with two levels of Fe supply. The soil was inoculated with rhizosphere microorganisms
only or with rhizosphere microorganisms together with an AM fungus ( Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe). An additional small soil compartment accessible to hyphae but not roots was added to
each pot after 6 weeks of plant growth. Radiolabelled P and Fe were supplied to the hyphae compartment 2 weeks after addition
of this compartment. After a further 2 weeks, plants were harvested and shoots were analysed for radiolabelled elements. In
both plant species, P uptake from the labelled soil increased significantly more in shoots of mycorrhizal plants than non-mycorrhizal
plants, thus confirming the well-known activity of the fungus in P uptake. Mycorrhizal inoculation had no significant influence
on the concentration of labelled Fe in shoots of peanut plants. In contrast, 59Fe increased in shoots of mycorrhizal sorghum plants. The uptake of Fe from labelled soil by sorghum was particularly high
under conditions producing a low Fe nutritional status of the plants. These results are preliminary evidence that hyphae of
an arbuscular mycorrhizal fungus can mobilise and/or take up Fe from soil and translocate it to the plant.
Accepted: 6 March 1998 相似文献
17.
Chicory plants ( Cichorium intybus L. var foliosum cv Flash) were tested with and without a 4-week-long cold treatment for in vivo and in vitro flowering potential every 2 weeks during the growing season. One hundred percent of the plants harvested 112 days or later
after sowing and then vernalized flowered in vivo. In vitro, no vernalization was needed to initiate flowering-stems on chicory explants taken from roots of 100 days old and older.
5-Azacytidine, a DNA demethylation agent, increased the flowering percentage on explants from young, vernalized roots but
could not induce more than 15% flowering on young, nonvernalized roots. The greater flowering potential of chicory root explants
in vitro when compared to plants of the same age tested in vivo was clearly established. This result suggests that some negative control on flowering was removed when root explants were
excised and the main plant body discarded.
Received: 31 August 1998 / Revision received: 27 October 1998 / Accepted: 10 November 1998 相似文献
18.
A member of the potato proteinase inhibitor II ( PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco ( Nicotiana tabacum) using Agrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R 0) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R 0 plants (designated R 1) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae of Chrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper), Spodoptera litura (F.) (Lepidoptera: Noctuidae) and Thysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently, C. eriosoma larvae fed leaf tissue from transgenic plants expressing the PPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectable PPI II protein accumulation. However, larvae of both S. litura and T. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured in C. eriosoma digestive tract extracts, but not in extracts from S. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants. 相似文献
19.
A simple method for the detection of DNA and RNA from small tissue samples (<-1 mg) is described. Stabs of fresh leaves from
wheat, clover, tobacco, broad bean, grape, tomato, lettuce, or asparagus were taken using glass microcapillaries, combined
with RNase inhibitor and subjected to RT-PCR either directly or after a DNase treatment. This method was used to successfully
detect the presence of (1) virus in infected plants, (2) transgenes such as neomycin phosphotransferase II in transformed plants and (3) various plant genes including RubiscoL and 1,3-β-D-glucanase. With the described DNase treatment,
the technique was shown to effectively detect RNA and, therefore, can be used to localize and study patterns of gene expression,
virus distribution, and/or transgene expression. The benefits of the described tissue stab technique are that it is quick,
it requires little manipulation, and is applicable to a variety of plants. 相似文献
20.
Studies of factors affecting host plant specialization by herbivores commonly highlight the value of the plant as both food
and habitat, but often cannot distinguish the relative importance of these plant traits. A different approach is to study
non-herbivorous animals that specialize on particular plants but do not feed on tissue from these plants. Such animals will
not be affected directly by the nutritional, chemical, or morphological traits that determine the value of the plant as a
food. This study reports on a filter-feeding amphipod, Ericthoniusbrasiliensis, that lives in domiciles it constructs by curling terminal segments of the green, calcified, and chemically defended seaweed
Halimedatuna. We examined the temporal (1850s–1990s) and spatial (Caribbean, Mediterranean, and Pacific regions) scale of the association,
the factors that may select for specialization on H. tuna, and the effect of the amphipod on growth of its host. Sampling along 125 km of coral reefs in the Florida Keys (USA) indicated
that almost all populations of H. tuna had been colonized by this amphipod. Infested plants occurred on nine of ten reefs that supported H. tuna populations, with between 8 and 75% of the plants on those reefs colonized by the amphipod. For infested plants, 2–23% of
all segments on each plant had been curled by the amphipod. Common co-occurring congeners of H. tuna ( H. opuntia and H. goreaui) were never used for domicile construction. A survey of 1498 Halimeda specimens collected during the last 140 years and archived in the U.S. National Museum of Natural History (Smithsonian Institution,
Washington, D.C.) indicated that the association has existed for >100 years and occurs throughout the Caribbean region, never
in the Indo-Pacific or Mediterranean, and only on H. tuna. Predation by fishes could select for amphipod specialization on H. tuna. Laboratory experiments demonstrated that amphipods inhabiting curled segments of H. tuna were relatively immune from fish predation while those on the exterior surface of the plant or in open water were rapidly
eaten. Segments of H. tuna are large enough to provide full protection from predators, while those of the co-occurring congeners H. goreaui and H. opuntia are of a size that may provide only partial protection. Experimental addition of E. brasiliensis to H. tuna plants in the field significantly decreased segment accumulation on infested relative to uninfested control plants. Whether
this negative effect was a direct or indirect consequence of amphipod occupancy is unclear. Rolling plant portions into domiciles
could directly decrease host growth by increasing shading and decreasing exposure of plant surface area to water column nutrient
flux. Amphipod occupancy could indirectly slow net host growth if fishes selectively feed on plant sections occupied by amphipods.
Underwater video showed that herbivorous fishes did not graze infested plants more than uninfested plants, but small predatory
fishes did prefer feeding from infested plants. These non-herbivorous fishes may slow host growth by damaging the terminal
meristematic tissues of plants during attacks on amphipods. This study demonstrates that habitat specialists can negatively
impact hosts without consuming them and that specialization on a plant can occur due to its habitat value alone (as opposed
to its value as a food).
Received: 24 March 1998 / Accepted: 1 November 1998 相似文献
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