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1.
Root segments from shoot tip-derived plantlets of the garlic (Allium sativum L.) clones `DDR7099', `PI383819', and `Piacenza' were utilized as an explant source for continuous, friable callus production.
The best callus production occurred on root segments initially cultured on medium with 4,5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 weeks, then subcultured to medium with 4.7 μm 4-amino-3,5,6-trichloropicolinic acid (picloram) +0.49 μm 6-(γ-γ-dimethylallylamino)purine (2iP) for 8 weeks. Embryogenic, friable callus was transferred to liquid medium for 1 month and
then transferred to solid regeneration medium for 14 weeks. The best shoot and root regeneration (85.3% and 35.8%, respectively)
occurred on 4-month-old calli from the clone `DDR7099'. In all clones, regeneration rate decreased as callus age increased.
Received: 14 October 1997 / Revision received: 26 December 1997 / Accepted: 12 January 1998 相似文献
2.
In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle
bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively
low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation
indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression.
Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997 相似文献
3.
Colin Charles Eady Carolyn Elizabeth Lister Yuying Suo David Schaper 《Plant cell reports》1996,15(12):958-962
Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient -glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient -glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.Abbreviations GUS
-glucuronidase
- IE
immature embryo
- MUG
methylumbelliferyl -D-glucuronide 相似文献
4.
Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells 总被引:7,自引:0,他引:7
This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium
scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient
expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated
tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS
activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings
the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently
deliver the plasmid DNA into intact plant cells.
Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998 相似文献
5.
Sergei F. Krasnyanski Jagdeep Sandhu Leslie L. Domier Dennis E. Buetow Schuyler S. Korban 《In vitro cellular & developmental biology. Plant》2001,37(4):427-433
Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa
mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their
effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable
regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation
of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed
the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic
plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative
tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression
of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than
when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio. 相似文献
6.
A. A. Watad D.-J. Yun T. Matsumoto X. Niu Y. Wu A. K. Kononowicz R. A. Bressan P. M. Hasegawa 《Plant cell reports》1998,17(4):262-267
We have obtained transgenic lily (Lilium longiflorum) plants after microprojectile bombardment, using the Biolistics PDS 1000/He system, of morphogenic calli derived from bulblet
scales, followed by bialaphos selection. Parameters which gave the highest transient uidA expression were used: a bombardment pressure of 1100 psi, a target distance of 6 cm and a 48-h preculture on medium with
3% sucrose. A total of 1800 morphogenic calli were co-bombarded with plasmids containing either the uidA reporter or PAT selectable marker genes. After bombardment, the calli were exposed to 2 mg/l bialaphos. Only 72 of the shoot-forming calli
(4%) survived. The 72 shoot clusters produced 342 shoots on elongation medium containing 0.5 mg/l bialaphos. Only 55 plantlets
survived subsequent exposure to 2.0 mg/l bialaphos. PCR analysis indicated that 19 of these plantlets contained the PAT transgene. Southern analysis of 3 of the plants indicated that all contained the PAT gene.
Received: 21 March 1997 / Revision received: 8 July 1997 / Accepted: 7 August 1997 相似文献
7.
Y. L. Nandadeva C. G. Lupi C. S. Meyer P. S. Devi I. Potrykus R. Bilang 《Plant cell reports》1999,18(6):500-504
Microprojectile-mediated transient and integrative transformation frequencies in rice (Oryza sativa cv. Taipei 309) embryogenic suspension cells were studied as a function of various parameters. Mannitol at concentrations
of 0.5 and 0.6 m was best for osmotic preconditioning of the cells for transient, but not for integrative transformation, for which sucrose
yielded the best and most reliable results. Denaturation of the transforming plasmid DNA prior to bombardment improved transient
and integrative transformation frequencies two to three fold. Delivery of double-stranded plasmids in linear form had no effect
on transient transformation when compared to supercoiled plasmid DNA, but led to an overall two fold increase in integrative
transformation frequency. This shows that optimized protocols for generating transgenic plants should not be based exclusively
on transient gene expression assays.
Received: 29 September 1997 / Revision received: 27 February 1998 / Accepted: 2 April 1998 相似文献
8.
Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the
Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in
both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the
gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression
in the clp -deficient mutant.
Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 相似文献
9.
10.
Somatic embryogenesis from mature leaves of rose (Rosa sp.) 总被引:9,自引:0,他引:9
Several plant growth regulators (0.3–53.3 μm 6-benzyladenine, 2,4-dichlorophenoxyacetic acid, gibberellic acid, 3-indoleacetic acid, p-chlorophenoxyacetic acid, kinetin and α-naphthylacetic acid), alone or in combination, and culture conditions were tested for their capacity to induce somatic embryogenesis
from mature leaf and stem explants of rose (Rosa sp.) of four commercial rose cultivars (Baccara, Mercedes, Ronto and Soraya). Somatic embryos were only induced from mature
leaf explants derived from Soraya on Murashige and Skoog (MS) medium supplemented with 53.5 μm
p-chlorophenoxyacetic acid and 4.6 μm kinetin, although satisfactory callus induction rates were obtained from all cultivars. After subculturing on the same medium,
embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto a MS medium
supplemented with 5.2 μm 6-benzyladenine and 5.7 μm 3-indoleacetic acid. Germination of mature embryos took place after subculturing them onto medium of the same composition.
Plantlets regenerated from embryos and bearing three to four leaves were transferred to a greenhouse.
Received: 4 February 1997 / Revision received: 28 August 1997 / Accepted: 1 October 1997 相似文献
11.
T. E. Gottschalk J. E. Nielsen P. Rasmussen 《Applied microbiology and biotechnology》1996,45(1-2):240-244
An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent,
when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values
between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH.
Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995 相似文献
12.
13.
Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts 总被引:1,自引:0,他引:1
A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described.
Protoplast yields of 3.0–5.0×106 were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10,
and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 105 protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified
KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation
was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed
after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus
formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium
containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal.
The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.
Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998 相似文献
14.
Sawahel WA 《Cellular & molecular biology letters》2002,7(1):49-59
The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene delivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical beta-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants. 相似文献
15.
Nathalie Leduc Victor Alejandro Iglesias Roland Bilang Andreas Gisel Ingo Potrykus Christof Sautter 《Sexual plant reproduction》1994,7(2):135-143
Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs. 相似文献
16.
Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium.
Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed.
Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997 相似文献
17.
Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures 总被引:1,自引:0,他引:1
The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production
from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced
on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18
μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the
culture treatments and, in general, low light intensities (50 μmol m–2 s–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart-
and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic
acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively.
Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997 相似文献
18.
A rapid and reliable micropropagation method was established for Spathoglottis plicata. Nodal and leaf explants dissected from 8-month-old pot-grown seedlings were cultured on charcoal-amended Murashige and Skoog
medium supplemented with 16 combinations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at concentrations
of 0.54–10.74 μm. Regeneration of protocorm-like bodies (PLBs) and subsequent plantlet development were observed from 98.5% of the nodal explants.
Only 6.5% of leaf explants and occasionally some root segments (dissected from regenerated plantlets) were able to produce
PLBs and then plantlets. The optimum plant growth regulator (PGR) combination for maximal PLB regeneration was 5.37 μm NAA and 0.44 μm BA. The best combination of PGR for plantlet development was 2.69–10.74 μm NAA and 8.88 μm BA. The NAA to BA ratios for maximal PLB induction and plantlet development were 12.2 and 0.3–1.2, respectively. Regenerated
PLBs and plantlets, when cut into pieces of less than 1 mm and subcultured onto the above media, regenerated new PLBs and
plantlets in another 3 months.
Received: 20 February 1997 / Revision received: 27 May 1997 / Accepted: 16 June 1997 相似文献
19.
This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7
mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic
acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture
of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt
solution with 7 mm CaCl2
.2H2O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength
MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were
cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to
shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred
to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks.
Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997 相似文献
20.
P. D. Needham R. G. Atkinson B. A. M. Morris R. C. Gardner A. P. Gleave 《Plant cell reports》1998,17(8):631-639
We have constructed a binary vector containing elements of the monopartite geminivirus, tobacco yellow dwarf virus (TYDV).
The vector is designed to be stably integrated into the plant genome, via Agrobacterium-mediated transfer. Upon expression of the viral replication-associated protein the TYDV elements are released from the T-DNA
and then replicate episomally. We refer to these released forms as multicopy plant episomes (MPE). Tobacco plants (Nicotiana tabacum cv `Samsun') transformed with the vector showed MPE release and subsequent episomal replication in 6 of 11 of these plants.
An MPE vector containing the uidA gene faithfully replicated and maintained the reporter gene, even though the construct was considerably larger than the wild-type
TYDV genome. Histochemical staining revealed a speckled GUS expression phenotype which could be correlated with MPE replication.
Received: 11 July 1997 / Revision received: 4 November 1997 / Accepted: 8 December 1997 相似文献