共查询到20条相似文献,搜索用时 31 毫秒
1.
Raham Sher Khan Syed Sartaj Alam Iqbal Munir Pejman Azadi Ikuo Nakamura Masahiro Mii 《Plant Cell, Tissue and Organ Culture》2011,106(1):11-20
The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue
and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system
has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In
an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium
tumefaciens strain EHA105. Infected leaf explants of P.
hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants
confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants
exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida. 相似文献
2.
The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by
the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one
of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs).
In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium
tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious
shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium
was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses
of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance
against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew). 相似文献
3.
The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous
presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation)
vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic
plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium
tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35
explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest
and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in
vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free
MS medium. 相似文献
4.
Hedayat Zakizadeh Henrik Lütken Sridevy Sriskandarajah Margrethe Serek Renate Müller 《Plant cell reports》2013,32(2):195-205
Key message
The P SAG12 -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions.Abstract
Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P SAG12 -ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P35S-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1–6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l?1) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants. 相似文献5.
Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt
−, GUS+ and excision+ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents. 相似文献
6.
MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic
plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant
growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious
shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately
4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1–3 for ipt gene and 1–2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium. 相似文献
7.
Xiang Liu Chun-Miao Feng Robert Franks Rongda Qu De-Yu Xie Qiu-Yun Jenny Xiang 《Plant cell reports》2013,32(1):77-87
Key message
Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.Abstract
Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants. 相似文献8.
Yoichi Ogawa Makoto Shirakawa Yasuko Koumoto Masaho Honda Yuki Asami Yasuhiro Kondo Ikuko Hara-Nishimura 《Plant cell reports》2014,33(7):1161-1172
Key Message
A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape.Abstract
Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the ‘herbicide dipping method’. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes. 相似文献9.
Key message
An efficient mannose selection system was established for transformation of Indica cultivar IR58025B . Different selection pressures were required to achieve optimum transformation frequency for different PMI selectable marker cassettes.Abstract
This study was conducted to establish an efficient transformation system for Indica rice, cultivar IR58025B. Four combinations of two promoters, rice Actin 1 and maize Ubiquitin 1, and two manA genes, native gene from E. coli (PMI-01) and synthetic maize codon-optimized gene (PMI-09) were compared under various concentrations of mannose. Different selection pressures were required for different gene cassettes to achieve corresponding optimum transformation frequency (TF). Higher TFs as 54 and 53 % were obtained when 5 g/L mannose was used for selection of prActin-PMI-01 cassette and 7.5 g/L mannose used for selection of prActin-PMI-09, respectively. TFs as 67 and 56 % were obtained when 7.5 and 15 g/L mannose were used for selection of prUbi-PMI-01 and prUbi-PMI-09, respectively. We conclude that higher TFs can be achieved for different gene cassettes when an optimum selection pressure is applied. By investigating the PMI expression level in transgenic calli and leaves, we found there was a significant positive correlation between the protein expression level and the optimal selection pressure. Higher optimal selection pressure is required for those constructs which confer higher expression of PMI protein. The single copy rate of those transgenic events for prActin-PMI-01 cassette is lower than that for other three cassettes. We speculate some of low copy events with low protein expression levels might not have been able to survive in the mannose selection. 相似文献10.
Xiuping Zou Aihong Peng Lanzhen Xu Xiaofeng Liu Tiangang Lei Lixiao Yao Yongrui He Shanchun Chen 《Plant cell reports》2013,32(10):1601-1613
Key message
A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.Abstract
The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants. 相似文献11.
Yurong Chen Anatoly Rivlin Andrea Lange Xudong Ye Zarir Vaghchhipawala Elizabeth Eisinger Erik Dersch Miriam Paris Brian Martinell Yuechun Wan 《Plant cell reports》2014,33(1):153-164
Key message
Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible.Abstract
We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7–8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology. 相似文献12.
A. Galambos A. Zok A. Kuczmog R. Oláh P. Putnoky W. Ream E. Szegedi 《Plant cell reports》2013,32(11):1751-1757
Key message
Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance.Abstract
Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. ‘Richter 110’ rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58. 相似文献13.
14.
Raham Sher Khan Gunaratnam Thirukkumaran Ikuo Nakamura Masahiro Mii 《Plant Cell, Tissue and Organ Culture》2010,101(3):279-285
The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem
for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies
to excise the selection marker gene from transgenic plants. Agrobacterium
tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free
half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated
and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine
(BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance,
short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of
DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only
LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing
rol-type MAT vector. 相似文献
15.
Guo-qing Song Aaron Walworth Dongyan Zhao Ning Jiang James F. Hancock 《Plant cell reports》2013,32(11):1759-1769
Key message
The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.Abstract
Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6–10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic ‘Aurora’ and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering. 相似文献16.
Thi Thu Huong Khuong Patrice Crété Christophe Robaglia Stefano Caffarri 《Plant cell reports》2013,32(9):1441-1454
Key message
An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions.Abstract
Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion. 相似文献17.
18.
Gunaratnam Thirukkumaran Raham Sher Khan Dong Poh Chin Ikuo Nakamura Masahiro Mii 《Plant Cell, Tissue and Organ Culture》2009,97(3):237-242
The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public
and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation
[MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium,
and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than
ninety percent of the normal shoots were ipt
−, gus
− but lacZ
+ as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting
perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene. 相似文献
19.
M. Narendran Satish G. Deole Satish Harkude Dattatray Shirale Asaram Nanote Pankaj Bihani Srinivas Parimi Bharat R. Char Usha B. Zehr 《Plant cell reports》2013,32(8):1191-1198
Key message
Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).Abstract
Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits. 相似文献20.