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1.
《Saudi Journal of Biological Sciences》2022,29(6):103292
Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l?1 in combination with 0.1 mg l?1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA3) (1.0 mg l?1), IAA (0.5 mg l?1), and ZEA (0.5 mg l?1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l?1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes. 相似文献
2.
参与在农杆菌介导遗传转化过程中的植物因子研究进展 总被引:5,自引:0,他引:5
随着农杆菌介导遗传转化过程中农杆菌一方转化机理的阐明,人们现在已经将目光转向了参与在农杆菌介导遗传转化过程中的植物因子。应用拟南芥突变体分析,酵母双杂交和cDNA的扩增片段长度多态性等技术人们已经部分阐明了参与在农杆菌向植物细胞的附着,TDNA的加工和转运,T链复合体向细胞核的转运,TDNA的整合等过程中的植物因子,并对植物基因对于农杆菌侵染的响应有了初步的了解。这些结果的获得不仅有助于人们加深对农杆菌介导遗传转化机理的认识而且有助于进一步扩大农杆菌介导遗传转化这一技术的应用范围 。 相似文献
3.
Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin,
inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation
duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant
to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD
deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the
transgenic tomato plants were resistant to 100 mg/l kanamycin.
Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284.
The text was submitted by the authors in English. 相似文献
4.
5.
The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene
transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous
efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all
the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears
to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce
stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of
the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general
guidelines for the development of a transformation protocol are discussed. 相似文献
6.
JoAnne J Fillatti James Sellmer Brent McCown Bruce Haissig Luca Comai 《Molecular & general genetics : MGG》1987,206(2):192-199
Summary A plant transformation and regeneration system has been developed for Populus species. Leaf explants, from stabilized shoot cultures of a Populus hybrid NC-5339 (Populus alba x grandidentata), were co-cultivated with Agrobacterium tumefaciens on a tobacco nurse culture. Both oncogenic and disarmed strains of A. tumefaciens harboring a binary vector which contained two neomycin phophotransferase II (NPT II) and one bacterial 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (aroA) chimeric gene fusions were used. Shoots did not develop when leaf explants were co-cultivated with the binary disarmed strain of A. tumefaciens. However, transformed plants with and without the wild type T-DNA were obtained using an oncogenic binary strain of A. tumefaciens. Successful genetic transformation was confirmed by NPT II enzyme activity assays, Southern blot analysis and immunological detection of bacterial EPSP synthase by Western blotting. This is the first report of a successful recovery of transformed plants of a forest tree and also the first record of insertion and expression of a foreign gene of agronomic importance into a woody plant species. 相似文献
7.
Miguel A. Providenti Hung Lee Jack T. Trevors 《Journal of industrial microbiology & biotechnology》1993,12(6):379-395
Summary The focus of this review is to examine some of the reasons biodegradation may not take place in the environment even though its occurrence in the laboratory has been demonstrated. Some approaches for dealing with chemical persistence will be discussed. In addition, the potential of bioremediation as an in situ clean-up technology will be considered. 相似文献
8.
Ticarcillin/potassium clavulanate is a very effective combination of antibiotics to eliminate Agrobacterium tumefaciens during tomato transformation. It shows no toxicity to tomato tissues at a concentration of 150 mg/l and significantly promotes
callus formation and shoot regeneration. The transformation frequency was raised more than 40% in comparison to cefotaxime.
Cefotaxime itself did not inhibit callus growth in culture medium, but it clearly decreased shoot differentiation. Together
with kanamycin, cefotaxime shows a strong negative effect on callus growth, shoot regeneration and transformation efficiency.
Unlike the widely used carbenicillin and cefotaxime, ticarcillin/potassium clavulanate is light stable and resistant to inactivation
by β-lactamase. Furthermore, ticarcillin/potassium clavulanate is more economical than carbenicillin and cefotaxime. In conclusion,
ticarcillin/potassium clavulanate is a very good alternative to eliminate Agrobacterium tumefaciens in plant transformation and has the potential to be widely used for plants which are sensitive to carbenicillin and cefotaxime.
Received: 22 September 1997 / Revision received: 7 November 1997 / Accepted: 15 December 1997 相似文献
9.
Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants 总被引:9,自引:0,他引:9
Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (β-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient
expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed
to selection 20 days after cocultivation. From 1419 original leaves, four shoots (A, B, C and D) were obtained that showed
amplification of the predicted DNA fragments by polymerase chain reaction (PCR). After micropropagation of these shoots, only
those cloned from shoot D gave consistently positive results in histochemical GUS detection and PCR amplification. Southern
blot hybridisation confirmed stable transgene integration in clone D, which was also negative in PCR amplification of an Agrobacterium gene. Additional molecular analysis suggested that the remaining three shoots (A, B and C) were chimeric.
Received: 28 March 1998 / Revision received: 18 April 1998 / Accepted: 12 May 1998 相似文献
10.
Carol Barrette Emily Cobb Ronald McNicol Gary Lyon 《Plant Cell, Tissue and Organ Culture》1997,47(2):135-144
Agrobacterium transformation systems forBrassica, Solanum andRubus, using carbenicillin, cefotaxime and ticaracillin respectively to eliminate contamination, were examined for the presence
of residualAgrobacterium. The results indicated that none of the antibiotics in question, succeeded in eliminatingAgrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent thatSolanum cultures senesced and died. This may be due to the fact that four times the Minimum bactericidal concentration values (concentration
to be used for elimination of contaminants in culture), for the three antibiotics, were higher than the concentrations employed
in the culture medium. Contamination in shoot material decreased over 16 to 24 weeks possibly due to bacteriostatis and the
use only of the apical node for further culture. The presence of the binary vector was also noted under non-selective conditions,
even up to 6 months after transformation, where approx. 50% of contaminated material still harboured bacterial cells with
the binary vector at levels of approx. 107 Colony forming units per gram. 相似文献
11.
An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar Blue Uniform was more responsive than White Uniform. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by -glucuronidase and ELISA analyses, respectively.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthalene acetic acid - TDZ Thidiazuron - BU Blue Uniform - WU White Uniform 相似文献
12.
Perumal Venkatachalam Natesan Geetha Narayanasamipillai Jayabalan Saravanababu Lakshmi Sita 《Journal of plant research》1998,111(4):565-572
Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants
followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot
regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before
transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting
transformation efficiency are discussed. 相似文献
13.
Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato 总被引:12,自引:0,他引:12
RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants. 相似文献
14.
Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for β-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and
leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of GUS activity in
plant tissues, and by Southern blotting analysis. In this transformation system, about 2 months were required for regeneration
of transgenic plants from stem and leaf segments. The frequency of transformation from stem segments was approximately 24%,
and the morphology of regenerated plants resembled that of the original parental strain.
Received: 2 September 1999 / Revision received: 30 November 1999 / Accepted: 4 December 1999 相似文献
15.
Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb) 总被引:1,自引:0,他引:1
Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin
or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium
containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that
the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed
shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months.
Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997 相似文献
16.
Activity of the CaMV 35S promoter in various parts of transgenic early flowering birch clones 总被引:3,自引:0,他引:3
J. Lemmetyinen K. Keinonen-Mettälä M. Lännenpää K. von Weissenberg T. Sopanen 《Plant cell reports》1998,18(3-4):243-248
Early flowering together with small size would be useful for various biotechnical or genetic studies on trees. We report
here the selection and micropropagation of early flowering birch (Betula pendula) clones (BPM1–12) obtained from seeds of birches bred elsewhere for early flowering. Under conditions that accelerate flowering
(a high CO2 level, strong and continuous illumination), the first male inflorescences emerged in 3–5 months, the trees then being 20–80
cm high. Transgenic lines (CaMV 35S-GUS INT) were produced through Agrobacterium-mediated gene transfer from BPM2, BPM5 and JR1/4 (a normally flowering birch). β-Glucuronidase (GUS) activities in the different lines, assayed 1–1.5 years after transformation, varied greatly. During further
in vitro culture for 10 months, the activities decreased to 0.3–7% of the original values. GUS activities were detected in
all organs studied, including the developing male inflorescences; the highest activity was in the roots.
Received: 28 April 1997 / Revision received: 5 September 1997 / Accepted: 30 November 1997 相似文献
17.
A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue 总被引:10,自引:1,他引:9
Caryl A. Chlan Junmin Lin Jeffery W. Cary Thomas E. Cleveland 《Plant Molecular Biology Reporter》1995,13(1):31-37
We have optimized methods for transformation of cotton meristem tissue using the Bio-Rad PDS/1000/He gene gun, selection of transformed tissue, and regeneration of transformed cotton plants. We have used either single or multiple bombardments of cotton tissue with 1.6-Å particles at rupture pressures of 90 or 110 kg/cm2. The distance between the tissue and the source of particles can be varied between 3 and 6 cm. After bombardment, transformed cotton tissue is identified by selection for growth on media supplemented with 50 μg/mL kanamycin. Tissue sections that form leaves, shoots and at least two roots are then transferred to media supplemented with 100 mg indoleacetic acid (IAA) to favor formation of extensive root systems. The plantlets are then transferred to soil, hardened off, and grown in the greenhouse. These plants have been confirmed to be transgenic by western-blot analysis of leaf protein extracts with polyclonal antiserum to the neomycin phosphotransferase II gene product. 相似文献
18.
Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS
ß-glucuronidase
- X-Gluc
5-bromo-4-chloro-3indolyl ß-D-glucuronic acid
- NPT II
neomycin phosphotransferase II 相似文献
19.
Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants 总被引:6,自引:0,他引:6
C.-K. Ho S.-H. Chang J.-Y. Tsay C.-J. Tsai V. L. Chiang Z.-Z. Chen 《Plant cell reports》1998,17(9):675-680
An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments
with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and
regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more
than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant
to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants.
Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis,
further confirming the integration and expression of T-DNA in these plants.
Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998 相似文献
20.
K. V. Krishnamurthy K. Suhasini A. P. Sagare M. Meixner A. de Kathen T. Pickardt O. Schieder 《Plant cell reports》2000,19(3):235-240
Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene
is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection
pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression
of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots
were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by
Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that
none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.
Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999 相似文献