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1.
Michal Moyal Ben Zvi Amir Zuker Marianna Ovadis Elena Shklarman Hagit Ben-Meir Shamir Zenvirt Alexander Vainstein 《Molecular breeding : new strategies in plant improvement》2008,22(4):543-553
As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility
of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment
of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA3-pretreated mother plants and a two-step selection scheme. The GA3 treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated
explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing
shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila
was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes
rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could
be beneficial to the ornamental industry. 相似文献
2.
An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA3 Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz 相似文献
3.
S. M. Touhidul Islam R. S. Tammi Sneh L. Singla-Pareek Zeba Islam Seraj 《Acta Physiologiae Plantarum》2010,32(4):657-663
Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for
improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na+/H+ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed.
Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type
plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than
wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive
stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield. 相似文献
4.
Jianzhong Lin Bo Zhou Yuanzhu Yang Jin Mei Xiaoying Zhao Xinhong Guo Xingqun Huang Dongying Tang Xuanming Liu 《Plant cell reports》2009,28(7):1065-1074
Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method
was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T
0) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T
0 plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation
was further confirmed by the genetic and molecular analyses of T
1 transformants. 相似文献
5.
Procedure for the Agrobacterium
tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue
(2 days) on medium supplemented with 100 μM acetosyringone, before bacterial infection significantly increased transient expression
of reporter gene (GUS). Co-cultivation period of 2 days and a bacterial density of 0.8 OD600 resulted in higher transient GUS expression. Method of injury to tissue, presence of acetosyringone in co-cultivation medium
and photoperiod during co-cultivation also influenced the expression of transient GUS activity. Amongst the three clones tested,
maximum transient GUS activity was recorded in clone ‘CE2’ followed by clone ‘T1’. Regeneration of transformed shoots was
achieved on modified Murashige and Skoog medium (potassium nitrate was replaced with 990 mg/l potassium sulphate and ammonium
nitrate with 392 mg/l ammonium sulphate, and mesoinositol concentration was increased to 200 mg/l). Stable transformation
was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to uidA and nptII genes. The absence of bacteria in the stable transformed tissues was confirmed by PCR amplification of fragment specific
to 16S rRNA of bacteria. 相似文献
6.
The physical barrier imposed by the thick microspore wall constitutes an obstacle for an efficient Agrobacterium-mediated transformation of vacuolate microspores prior to androgenic induction and haploid embryogenic commitment. It is
thus necessary to implement additional methods to overcome this drawback. In this study, we focused on the optimization of
a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner. We tested different options, based
on microprojectile bombardment, to be applied prior to agroinfiltration. From them, the best results were obtained through
co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by Agrobacterium tumefaciens infection. This method provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis
induction. 相似文献
7.
Plants are sensitive to D-serine, but functional expression of the dsdA gene, encoding D-serine ammonia lyase, from Escherichia coli can alleviate this toxicity. Plants, in contrast to many other organisms, lack the common pathway for oxidative deamination of D-amino acids. This difference in metabolism has major consequences for plant responses to D-amino acids, since several D-amino acids are toxic to plants even at relatively low concentrations. Therefore, introducing an enzyme specific for a phytotoxic D-amino acid should generate a selectable characteristic that can be screened. Here we present the use of the dsdA gene as a selectable marker for transformation of Arabidopsis. D-serine ammonia lyase catalyses the deamination of D-serine into pyruvate, water and ammonium. dsdA transgenic seedlings can be clearly distinguished from wild type, having an unambiguous phenotype immediately following germination when selected on D-serine containing medium. The dsdA marker allows flexibility in application of the selective agent: it can be applied in sterile plates, in foliar sprays or in liquid culture. Selection with D-serine resistance was compared with selection based on kanamycin resistance, and was found to generate similar transformation frequencies but also to be more unambiguous, more rapid and more versatile with respect to the way the selective agent can be supplied. 相似文献
8.
Indrajit Dutta Prasenjit Saha Sampa Das 《In vitro cellular & developmental biology. Plant》2008,44(5):401-411
Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige
and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver
nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency
of stable transformation was found to be approximately 19% in the T
0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern
blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of
T
1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency
over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea. 相似文献
9.
A protocol was developed for Agrobacterium-mediated genetic transformation of Acacia mangium using rejuvenated shoots as the explant. Axillary buds and shoot apices of adult trees were rejuvenated by culturing them on Murashige and Skoog (MS) medium, and stem segments of rejuvenated shoots were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI121. The selection for transgenic shoots was performed through five consecutive steps on MS medium supplemented with 1.0 mg/l thidiazuron, 0.25 mg/l indole-3-acetic acid and different concentrations of geneticin (G418; 12–30 mg/l) and timentin (T; 50–300 mg/l) in the following order: 12 mg/l G418 and 300 mg/l T for 30 days, 20 mg/l G418 and 200 mg/l T for 60 days, 30 mg/l G418 and 100 mg/l T for 30 days, 12 mg/l G418 and 50 mg/l T for 30 days, and finally 15 mg/l G418 and 5 mg/l gibberellic acid (GA3) for 60 days. Thirty-four percent of the stem segments produced resistant multiple adventitious shoot buds, of which 30% expressed the β-glucuronidase gene. The shoot buds were subjected to repeated selection on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine, 2.5 mg/l GA3 and 20 mg/l G418. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 2.0 mg/l α-naphthaleneacetic acid, 0.1 mg/l kinetin and 20 mg/l G418. Genomic Southern blot hybridization confirmed the incorporation of the NPTII gene into the host genome. 相似文献
10.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
11.
Yang Jiang Lin Ling Lingli Zhang Abigail Domingo Mingli Cai Chengfang Li Ming Zhan Jinping Wang Cougui Cao 《Acta Physiologiae Plantarum》2017,39(3):89
Transgenic Bacillus thuringiensis (Bt) rice have been reported to acquire effective resistance against the target pests; however, the insertion and expression of alien Bt genes may have some unintended effects on the growth characteristics of rice. A screen-house experiment was conducted and repeated twice to investigate the growth characteristics and Bt protein expressions in two Bt rice lines [MH63 (Cry2A*) and MH63 (Cry1Ab/Ac)], which had different Bt protein expression levels in leaves, under zero nitrogen (N0) and recommended nitrogen (NR) fertilizer applications. Compared to the counterpart MH63, MH63 (Cry2A*) under N0 experienced accelerated leaf senescence and a lower internal N use efficiency (IEN), resulting in a 23.2% decrease in grain yield and a lower accumulated biomass. These variations were revealed to be correlated to the higher ratio of the Bt protein content to the soluble protein content (BTC/SPC) with a maximum value of 4.3‰ in MH63 (Cry2A*) leaves in the late growth stage. Under NR, no differences in growth characteristics between MH63 (Cry2A*) and MH63 were found. The growth characteristics of MH63 (Cry1Ab/Ac), with a lower BTC/SPC in the late growth stage compared to MH63 (Cry2A*), were identical to those of MH63 under the two N applications. Results show that the transgenic Bt rice MH63 (Cry2A*), with a relatively higher Bt protein expression in the late growth stage, had an inferior adaptation to nitrogen deficiency compared to its non-Bt counterpart. And this inferior adaptation was found to be correlated with the higher BTC/SPC in MH63 (Cry2A*) leaves in the late growth stage. 相似文献
12.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
13.
14.
Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were
obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT
(2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern
analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants
followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency
as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew
on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization. 相似文献
15.
As a first step in the development of a successful Agrobacterium tumefaciens mediated transformation method for kenaf, factors influencing the successful T-DNA integration and expression (as measured by the GUS expression) were investigated. Transformation was carried out using two kenaf cultivars and Agrobacterium strain EHA 105 carrying different vectors, plasmid pIG 121-Hm or pEC:gus. Pre-culturing the explants for 2days in benzyl adenine containing medium, and wounding the explant before inoculation were found to enhance the transient GUS expression. Increasing the duration of pre-culture and co-culture period enhanced the transient GUS expression up to a threshold level. Increased transient GUS expression did not correlate with an increase in stable expression. Gene integration was confirmed by PCR analysis. 相似文献
16.
M. Srinivas Prasad B. Aruna Kanthi S. M. Balachandran M. Seshumadhav K. Madhan Mohan B. C. Viraktamath 《World journal of microbiology & biotechnology》2009,25(10):1765-1769
Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza
sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including
rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation
ratio of 3:1 for resistant to susceptible F2 plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001. 相似文献
17.
Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained
by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5′-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from
Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated
gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method. 相似文献
18.
Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species. 相似文献
19.
Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report
the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup
helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very
few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the
segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the
first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant. 相似文献
20.
Wissam A. Abou-Alaiwi Shobha D. Potlakayala Stephen L. Goldman Puthiyaparambil C. Josekutty Deepkamal N. Karelia Sairam V. Rudrabhatla 《Plant Cell, Tissue and Organ Culture》2012,109(1):1-8
An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally
regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter.
A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including
PCR, Southern blotting and RT-PCR, were performed on T0 and T1 plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants.
Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay. 相似文献