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1.
Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler 总被引:4,自引:0,他引:4
Barceló Marta El-Mansouri Iman Mercado José A. Quesada Miguel A. Pliego Alfaro Fernando 《Plant Cell, Tissue and Organ Culture》1998,54(1):29-36
The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse
grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM
IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk.
Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The
regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was
attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in
the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance
after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed
by the GUS assay and PCR analysis.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
2.
White Kwao Krua variety classification by botanical characteristics and ISSR-Touchdown PCR technique
White Kwao Krua [Pueraria candollei Grah. var. mirifica (Airy Shaw et Suvatabandhu) Niyomdham], is a herb used as an ingredient in supplementary and cosmetic. The tuberous roots
of White Kwao Krua (WKK) contain estrogen-like substances. Seeds of WKK, collected from Prachuab Khiri Khan, were planted
and propagated in the farm of Suranaree University of Technology, and their genetic backgrounds were ambiguous. Thirty six
plants of WKK in the same age were sampled for classification using 7 botanical characteristics and DNA fingerprint by ISSR-Touchdown
PCR technique. The relationship of the 7 botanical characteristics, using principle component analysis (PCA), showed the WKK
plants fell into 3 groups. In the first group was plant number 34, which was distinguished from the other plants by its small
leaf size. The second group consisted of 23 plants with elliptic leaf shape, acute leaf base, and acuminate leaf apex. The
third group consisted of 12 plants with ovate leaf shape, obtuse leaf base, and cuspidate leaf apex. The ISSR-Touchdown PCR
technique with 41 primers detected 355 loci of DNA with an average of 8.6 loci per primer. The sizes of DNA ranged between
280 bp to 1550 bp. Two hundred ninety three loci exhibited polymorphisms (82.54%) and the rest 62 loci were monomorphic (17.46%).
The polymorphism information content (PIC) was between 0.0315–0.9779 (average 0.4779) and number of effective alleles per
locus (N
e) ranged between 1.1250–1.8541 (average 1.5544). Unweighted pair group method with arithmetic mean (UPGMA), Jaccard similarity
coefficient and PCA were used to find the construction of genetic relationship of WKK. The genetic similarity (GS) of WKK
ranged between 0.50–0.86 (average 0.77). At the GS of 0.56 from cluster analysis, the WKK varieties could be divided in 2
major groups. The first group comprised of plant number 34 and 7, and the second group could be further divided into 2 subgroups
at GS of 0.69. None of the WKK plants was identical in genetic, and they were expected to be derived from 5 genetic sources.
These results showed that applying of the ISSR-Touchdown PCR technique could be used to classify WKK efficiently. 相似文献
3.
Yu-Ji Lian Guang-Zhe Lin Xiao-Mei Zhao Hak-Tae Lim 《In vitro cellular & developmental biology. Plant》2011,47(2):289-296
Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf
mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid,
0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum.
At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon
source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d,
when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l
indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed
a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase
chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic
DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed. 相似文献
4.
Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants 总被引:1,自引:0,他引:1
Amjad Masood Husaini 《Plant cell reports》2010,29(1):97-110
Following previously described Agrobacterium tumefaciens-mediated transformation procedures for Fragaria × ananassa Duch. ‘Chandler’, we undertook several experiments to establish the importance of some parameters affecting transformation.
The most important factor that increased the percent recovery of transformants was the introduction of a pre-selection phase,
in-between co-cultivation and selection, in which leaf disks were cultured on pre-selection regeneration medium containing
validamycin A, timentin, and cefotaxime. The average percentage of leaf disks forming shoots on selection medium containing
cefotaxime (250 mg l−1) + timentin (250 mg l−1) was 5.4% and about three shoots per regenerating leaf disk. Maximum transformation percentage, based on polymerase chain
reaction, was 31.25%. Transgene integration and copy number were assessed by Southern hybridization confirming single copy
as well as multiple copies of transgene integration in shoots as well as roots separately. This confirmed the non-chimeric
nature of these transgenic plants. The system is very promising for the regeneration of genetically transformed cells and
obtaining transgenic strawberry plants at high efficiency. 相似文献
5.
Natural isothiocyanates, produced during plant tissue damage from methionine-derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell-free leaf extracts as inducers of murine QR, which reduces samples preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine-derived glucosinolates, as shown by the analysis of wild-type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate-based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate-producing species. 相似文献
6.
Chirita luochengensis, a new species of Gesneriaceae from Guangxi, China, is described and illustrated. The new species is similar to C. linearifolia in leaf shape, but can be distinguished by the apex of leaf blade obtuse to round, corolla purplish, 3–4.2 cm long, corolla
tube 2–2.5 cm long, 10–15 mm diameter at the mouth, staminodes 3, disc ca. 2.5 mm in height, pistil 2.5–3 cm long, stigma
obtrapeziform, 4 mm long, and apex shallowly 2-lobed. 相似文献
7.
High-quality plant DNA extraction for PCR: an easy approach 总被引:1,自引:0,他引:1
I. Ahmed M. Islam W. Arshad A. Mannan W. Ahmad B. Mirza 《Journal of applied genetics》2009,50(2):105-107
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies.
For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and
efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires
maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes,
followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether
is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm−2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR
using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers
by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato
and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability
of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive. 相似文献
8.
9.
The ecophysiological linkage of leaf phosphorus (P) to photosynthetic capacity (A
max) and to the A
max–nitrogen relation remains poorly understood. To address this issue we compiled published and unpublished field data for mass-based
A
max, nitrogen (N) and P (n = 517 observations) from 314 species at 42 sites in 14 countries. Data were from four biomes: arctic, cold temperate, subtropical
(including Mediterranean), and tropical. We asked whether plants with low P levels have low A
max, a shallower slope of the A
max–N relationship, and whether these patterns have a geographic signature. On average, leaf P was substantially lower in the
two warmer than in the two colder biomes, with the reverse true for N:P ratios. The evidence indicates that the response of
A
max to leaf N is constrained by low leaf P. Using a full factorial model for all data, A
max was related to leaf N, but not to leaf P on its own, with a significant leaf N × leaf P interaction indicating that the
response of A
max to N increased with increasing leaf P. This was also found in analyses using one value per species per site, or by comparing
only angiosperms or only woody plants. Additionally, the slope of the A
max–N relationship was higher in the colder arctic and temperate than warmer tropical and subtropical biomes. Sorting data into
low, medium, and high leaf P groupings also showed that the A
max–N slope increases with leaf P. These analyses support claims that in P-limited ecosystems the A
max–N relationship may be constrained by low P, and are consistent with laboratory studies that show P-deficient plants have
limited ribulose-1,5-bisphosphate regeneration, a likely mechanism for the P influence upon the A
max–N relation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
Osvaldo A. Castellanos-Hernández Araceli Rodríguez-Sahagún Gustavo J. Acevedo-Hernández Benjamín Rodríguez-Garay José Luis Cabrera-Ponce Luis Rafael Herrera-Estrella 《Plant Cell, Tissue and Organ Culture》2009,99(2):175-181
A biolistic protocol for the stable genetic transformation of the hardwood tree Paulownia elongata was developed. Leaf explants were bombarded using the PDS-1000/He system with plasmid pBI121. The introduced DNA contained
the β-glucuronidase (GUS) reporter gene and neomycin phosphotransferase (nptII) as a selection marker. Transformed calli were induced and selected on medium supplemented with 50 mg L−1 kanamycin, and transgenic plants were regenerated through indirect organogenesis. Complete plants were successfully transferred
to soil and established under greenhouse conditions. Different helium pressures and explant positions were used and the transformation
frequency was calculated. Optimal conditions for genetic transformation were bombardment of the abaxial leaf surface at a
pressure of 450 psi. The integration of the transgenes in the plant genome and their stable expression was demonstrated by
fluorometric GUS assay, determination of NPTII activity and PCR analysis. This method allows the production of transgenic
trees of P. elongata in a relatively short time. 相似文献
11.
Mark D. Burow Caryl A. Chlan Partha Sen Andreana Lisca Norimoto Murai 《Plant Molecular Biology Reporter》1990,8(2):124-139
A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants. Modifications include transformation ofAgrobacterium by a freeze-thaw procedure, initial cocultivation of leaf disks andAgrobacterium under vacuum, subsequent growth with nurse cells for one week, rooting of shoots in medium lacking carbenicillin, longer,
growth in rooting medium, and a shortened “hardening” step. By this procedure, an average of 1.3 kanamycin-resistant calli
were obtained per leaf disk, and 38% of, the callus cultures used were regenerated to produce 133 independently transformed
tobacco plants. 相似文献
12.
番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用 总被引:4,自引:0,他引:4
以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。 相似文献
13.
Genotypic variation in drought response of silver birch (Betula pendula Roth): leaf and root morphology and carbon partitioning 总被引:1,自引:0,他引:1
This study investigates the drought response of four genotypes of Betula pendula with a focus on leaf and root morphological traits, leaf phenology and carbon partitioning between shoot and root. Potted
one-year-old clonal plants of four genotypes from regions with low to high annual rainfall (550–1270 mm year−1) were subjected to drought periods of 12–14 weeks in two subsequent years. Well-watered control plants of the four genotypes
differed significantly with respect to total leaf area per plant (LA) and specific leaf area (SLA), whereas differences in
total fine root surface area (RA), root specific area (SRA), and the fine root:leaf mass ratio (FR:LM) were not significant.
Highest LA and SLA were found in the clone originating from the driest environment. In complementary physiological investigations
this clone was found to have the highest water use as well which was interpreted as competitive superiority in terms of water
consumption. Drought resulted in an increase in SLA in all genotypes, and a decrease in LA. Leaf area reduction was more pronounced
in the genotypes from high than in those from low rainfall origin. The ratio of total root to leaf surfaces remained more
or less constant after drought application despite an increase in FR:LM. This is explained by a decrease in SRA resulting
from a reduced abundance of very small fine rootlets (diameter <0.2 mm) in the drought-treated plants. The loss in total root
surface area due to a reduction in finest root mass was compensated for by a relative increase in total root dry mass per
plant. Comparison of results from the first and second drought period indicated a marked influence of timing of drought, root
system size, and putative root limitation on plant drought response. We conclude that leaf and root morphology, the total
leaf and root surfaces, and the morphological response to drought in birch are to a large extent under genetic control. 相似文献
14.
Tetraploid plants were produced from leaf explants of diploid Populus pseudo-simonii by treating the leaves with colchicine. Leaf explants were cultured on MS basal medium containing 1.78 μM BA and 1.08 μM
NAA for 0, 6 and 12 days, and then transferred to the same MS liquid medium with colchicine at concentrations of 25, 50 and
75 μM for 1, 2 and 3 days. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that were pre-cultured
for 6 days and then cultured in liquid MS with 50 μM colchicine for 3 days. Flow cytometric analysis was used to screen the
tetraploids out from the regenerated plants and chromosome number counting was employed to confirm the polyploidy level. Size
and frequency of leaf stomata between diploid and tetraploid plants were demonstrated to have significant differences. 相似文献
15.
Athanasios Tsaftaris Konstantinos Pasentzis Anagnostis Argiriou 《Biotechnology letters》2010,32(1):157-161
We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse
PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification
of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the
circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse
PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic
sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different
species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production. 相似文献
16.
Diwakar Aggarwal Anil Kumar M. Sudhakara Reddy 《Plant Cell, Tissue and Organ Culture》2010,102(1):45-52
An efficient shoot organogenesis system has been developed from mature plants of selected elite clones of Eucalyptus tereticornis Sm. Cultures were established using nodal explants taken from freshly coppice shoots cultured on Murashige and Skoog medium
containing 58 mM sucrose, 0.7% (w/v) agar (MS medium) and supplemented with 2.5 μM benzyladenine (BA) and 0.5 μM α-naphthaleneacetic
acid (NAA). Shoot organogenesis was achieved from leaf segments taken from elongated microshoots on MS medium supplemented
with 5.0 μM BA and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of cefotaxime to the medium promoted shoot
differentiation, whereas carbenicillin and cephalexin inhibited shoot differentiation. Maximum shoot bud organogenesis (44.6%)
occurred in explants cultured on MS medium supplemented with 5.0 μM BA, 1.0 μM 2,4-D and 500 mg/l cefotaxime. Leaf maturity
influenced shoot regeneration, with maximum shoot organogeneisis (40.5%) occurring when the source of explants was the fifth
leaf (14–16 days old) from the top of microshoot. Shoot organogenic potential also varied amongst the different clones of
E. tereticornis. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses indicated clonal uniformity of
the newly formed shoots/plants, and these were also found to be true-to-type. 相似文献
17.
We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted
chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin
formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm
spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar
short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they
were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed
that the 30–40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends
uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on
their topology and high degree of interconnectedness, it is unlikely that 30–40-nm domains arise from the folding of local
stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments.
By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network,
while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30–40-nm chromatin
domains. These observations suggest that 30–40-nm domains are essential for establishing long-range chromatin associations
that are central for chromosome condensation.
Electronic supplementary material The online version of this article (doi ) contains supplementary material, which is available to authorized users. 相似文献
18.
Awadhesh Kumar Arya Kottackal Poulose Martin Aneta Sabovljevic Joseph Madassery 《Acta Physiologiae Plantarum》2011,33(5):2011-2017
A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A4M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A4M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection
with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal
medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious
roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and
PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A4M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots. 相似文献
19.
Abolghasem Abbasi Kejani Sayed Ali Hosseini Tafreshi Sayed Mojtaba Khayyam Nekouei Mohammad Reza Mofid 《Molecular biology reports》2010,37(2):797-800
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew
(Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation
were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform
extraction steps from the isolation procedure. Both spectrophotometric (A260/A280 and A260/A230 ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the
average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g
leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. 相似文献
20.
Andrea Menéndez-Yuffá Dominique Barry-Etienne Benoît Bertrand Frédéric Georget Hervé Etienne 《Plant Cell, Tissue and Organ Culture》2010,102(3):297-307
Plants of Coffea arabica L. derived via somatic embryogenesis, namely, somaclones, were evaluated with C. arabica seedlings grown in the nursery. At the time of their transfer to the nursery, somaclones of C. arabica cvs. Caturra and Costa Rica 95 (Catimor) were smaller and less vigorous than seedlings of the same cultivars. Following an
initial slow growth for a period of 10 weeks, somaclones began to grow faster than seedlings until both groups of plants were
equal in size at 21 weeks (entire duration of growth in the nursery). Comparisons of aerial and root systems of 30-cm long
somaclones and seedlings of two cultivars revealed that plants of somaclones were more vigorous than seedlings, based on the
higher number of leaves (13–16 vs. 9), larger leaf area (1060–1280 vs. 730–890 cm2), and greater dry weight of aerial organs (8.5–12 vs. 7.0–7.5 g). For cv. Caturra, the root dry weight of somaclones was
significantly greater than that of seedlings (2.7 vs. 1.9 g) and was attributable to the large diameter roots (>0.5 mm).
Analysis of 176,000 F1 hybrid somaclones revealed that these exhibited more heterogeneous growth than did the seedlings derived from zygotic embryos;
moreover, there was a genotype effect. Almost 9–20% of somaclones required an additional 3–4 months of growth in the nursery,
and 8–12% were culled for other undesirable horticultural attributes. Only 0.10–0.23% of somaclones displayed variant phenotypes.
The observed somaclone vigor in the nursery was carried over to field performance as these plants were more precocious than
seedlings and yielded coffee beans 1 year earlier than seedlings. 相似文献