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1.
3 Biotech ; 9(5): 192, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31065492

RESUMO

Natural antimicrobial peptides have been shown as one of the important tools to combat certain pathogens and play important role as a part of innate immune system in plants and, also adaptive immunity in animals. Defensin is one of the antimicrobial peptides with a diverse nature of mechanism against different pathogens like viruses, bacteria and fungi. They have a broad function in humans, vertebrates, invertebrates, insects, and plants. Plant defensins primarily interact with membrane lipids for their biological activity. Several antimicrobial peptides (AMPs) have been overexpressed in plants for enhanced disease protection. The plants defensin peptides have been efficiently employed as an effective strategy for control of diseases in plants. They can be successfully integrated in plants genome along with some other peptide genes in order to produce transgenic crops for enhanced disease resistance. This review summarizes plant defensins, their expression in plants and enhanced disease resistance potential against phytopathogens.

2.
Sci Rep ; 8(1): 16556, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410086

RESUMO

The application of fluorescent proteins in ornamental plants has lagged behind despite the recent development of powerful genetic tools. Although we previously generated transgenic torenia plants expressing green fluorescent protein from marine plankton (CpYGFP), in which bright fluorescence was easily visible at the whole plant level, the maximum excitation of this protein within the visible light spectrum required the use of a coloured emission filter to eliminate exciting light. Here, to overcome this limitation, we generated transgenic petunia plants expressing eYGFPuv, a CpYGFP derivative exhibiting bright fluorescence under invisible ultraviolet (UV) light excitation, with a novel combination of transcriptional terminator plus translational enhancer. As expected, all transgenic plants exhibited brilliant green fluorescence easily visible to the naked eye without an emission filter. In addition, fluorescence expressed in transgenic petunia flowers was stable during long-term vegetative propagation. Finally, we visually and quantitatively confirmed that transgenic petunia flowers resist to long-term exposure of UV without any damages such as fluorescence decay and withering. Thus, our whole-plant fluorescence imaging tool, that does not require high sensitive imaging equipment or special imaging conditions for observation, might be useful not only for basic plant research but also for ornamental purposes as a novel flower property.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Petunia/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Elementos Facilitadores Genéticos , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Petunia/crescimento & desenvolvimento , Petunia/metabolismo , Plâncton/genética , Plâncton/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Compostos de Amônio Quaternário , Raios Ultravioleta
3.
Breed Sci ; 68(3): 360-366, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30100803

RESUMO

Lilies (Lilium spp.) are one of the most important floricultural crops. As most lily cultivars have originated from interspecific hybridization, they usually have complex genome composition and occasionally fail to develop normal gametes. Further improvement of lily cultivars by sexual crossing requires evaluation of gamete development and subsequent male and female fertility. Although male fertility is easily evaluated through microscopic observation after staining or by pollen culture for germination, evaluation of female fertility is difficult, because gametes develop inside an ovule within an ovary. Lilium species have the Fritillaria type of embryo sac, which, at maturity, consists of a haploid egg apparatus, including one haploid egg cell and two haploid synergids, two polar nuclei (one haploid nucleus and one triploid nucleus) and three triploid antipodal cells. Compared to the Polygonum type of embryo sac, composition of the embryo in the Fritillaria type of embryo sac is complex. We developed an efficient microscopic observation technique for ovules using the clearing procedure, which allowed us to categorize abnormal patterns of female gametes and to elucidate the frequency of abnormal female gamete development. The relationship among normal embryo sac, pollen stainability and seed formation in lily cultivars is discussed.

4.
Physiol Mol Biol Plants ; 24(3): 503-511, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29692557

RESUMO

Rose cultivars with blue flower color are among the most attractive breeding targets in floriculture. However, they are difficult to produce due to the low efficiency of transformation systems, interactive effects of hosts and vectors, and lengthy processes. In this study, agroinfiltration-mediated transient expression was investigated as a tool to assess the function of flower color genes and to determine appropriate host cultivars for stable transformation in Rosa hybrida. To induce delphinidin accumulation and consequently to produce blue hue, the petals of 30 rose cultivars were infiltrated with three different expression vectors namely pBIH-35S-CcF3'5'H, pBIH-35S-Del2 and pBIH-35S-Del8, harbouring different sets of flower color genes. The results obtained showed that the ectopic expression of the genes was only detected in three cultivars with dark pink petals (i.e. 'Purple power', 'High & Mora' and 'Marina') after 6-8 days. The high performance liquid chromatography analyses confirmed delphinidin accumulation in the infiltrated petals caused by transient expression of CcF3'5'H gene. Moreover, there were significant differences in the amounts of delphinidin among the three cultivars infiltrated with the three different expression vectors. More specifically, the highest delphinidin content was detected in the cultivar 'Purple power' (4.67 µg g-1 FW), infiltrated with the pBIH-35S-Del2 vector. The expression of CcF3'5'H gene in the infiltrated petals was also confirmed by real time PCR. In conclusion and based on the findings of the present study, the agroinfiltration could be regarded as a reliable method to identify suitable rose cultivars in blue rose flower production programs.

5.
Plant Biotechnol (Tokyo) ; 33(4): 297-307, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-31274991

RESUMO

In Arabidopsis, flowering is delayed under red light and induced under far red light and blue light. Studies suggest that the florigen, FLOWERING LOCUS T, is involved in the control of light quality-associated flowering in Arabidopsis. In petunia, similar to Arabidopsis, flowering is delayed under red light and induced under blue light, however its mechanism still remains unknown. Here we isolated a gene which has 75% amino acid sequence similarity with Arabidopsis FT (AtFT), named PehFT. By overexpressing PehFT in Arbidopsis and petunia, we tested its ability to induce flowering. Also, by conducting expression analyses of PehFT under different light quality treatments, we tested its response to light quality. We concluded that PehFT, like AtFT, is a gene which responds to photoperiod and light quality, but unlike AtFT, is not the main gene controlling the light quality-associated flowering.

6.
PLoS One ; 10(4): e0120551, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25901740

RESUMO

Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.


Assuntos
Begomovirus/genética , Engenharia Genética , Manihot/virologia , Vírus do Mosaico/genética , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/virologia , RNA Interferente Pequeno/genética , DNA Viral/genética , Manihot/genética , Manihot/crescimento & desenvolvimento , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/virologia , Reação em Cadeia da Polimerase
7.
Breed Sci ; 65(5): 396-402, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26719742

RESUMO

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO3. Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.

8.
J Nat Med ; 68(4): 677-85, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24952707

RESUMO

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.


Assuntos
Citometria de Fluxo , Papaver/classificação , DNA de Cloroplastos/química , Flores/química , Japão , Entorpecentes/análise , Papaver/anatomia & histologia , Papaver/química , Papaver/genética , Tebaína/análise
9.
Mol Biotechnol ; 56(9): 814-23, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24802621

RESUMO

Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.


Assuntos
Alternaria/patogenicidade , Quitinases/metabolismo , Defensinas/metabolismo , Fusarium/patogenicidade , Plantas Geneticamente Modificadas/microbiologia , Solanum tuberosum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/genética , Defensinas/genética , Técnicas In Vitro , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Solanum tuberosum/microbiologia , Streptomyces griseus/enzimologia , Transformação Genética , Wasabia/metabolismo
10.
Plant Cell Rep ; 33(3): 411-21, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24311155

RESUMO

KEY MESSAGE: Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker. ABSTRACT: Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin 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 cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.


Assuntos
Alternaria/patogenicidade , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Solanum melongena/metabolismo , Solanum melongena/microbiologia , Wasabia/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Recombinação Genética/genética , Solanum melongena/genética
11.
Mol Biotechnol ; 56(1): 50-63, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23820979

RESUMO

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909-1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.


Assuntos
Agrobacterium tumefaciens/genética , Cucumovirus/enzimologia , Folhas de Planta/virologia , RNA Replicase/genética , RNA Replicase/metabolismo , Tabaco/virologia , Proteínas Virais/metabolismo , Agrobacterium tumefaciens/metabolismo , Cucumovirus/genética , Genes Virais , Sequências Repetidas Invertidas , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Deleção de Sequência , Tabaco/genética , Proteínas Virais/genética
12.
Transgenic Res ; 22(6): 1191-205, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23748933

RESUMO

Cucumber mosaic virus is an important plant pathogen with a broad host range encompassing many plant species. This study demonstrates the production of transgenic potato lines exhibiting complete resistance to cucumber mosaic virus strain O and Y by post transcriptional gene silencing. Two constructs were used, one, pEKH2IN2CMVai, contains inverted repeat of 1,138 bp fragment of a defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O), while the other, TRV-based VIGS vector (pTRV2CMVai), contains the same fragment of the replicase gene, but without inverted repeat. These constructs were used to produce transgenic potato lines of cultivar 'Danshaku', a susceptible genotype to CMV. Transgenic lines derived from pEKH2IN2CMVai accumulated small interfering RNA (siRNA) before and after virus challenge, whereas those derived from pTRV2CMVai showed siRNA expression after virus challenge. When transgenic lines were challenged with CMV-O or CMV-Y, four lines exhibited complete (100%) resistance to both strains, whereas the other lines had high levels of resistance. Infectivity of CMV-O was lower than that of CMV-Y in the highly resistant plants. There were no significant differences with regard to resistance between plants derived from pEKH2IN2CMVai and those obtained from pTRV2CMVai. The presence of CMV-specific siRNA in the resistant phenotypes indicates that the resistance was acquired through RNA silencing.


Assuntos
Cucumovirus/patogenicidade , Plantas Geneticamente Modificadas/genética , RNA Replicase/genética , Solanum tuberosum/genética , Cucumovirus/genética , Resistência à Doença/genética , Inativação Gênica , Doenças das Plantas/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Solanum tuberosum/virologia
13.
Breed Sci ; 62(2): 113-23, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23136522

RESUMO

Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. 'Orange Rubra' (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l(-1) α-naphthylacetic acid, 0.1 mg l(-1) 6-benzyladenine, 10 mg l(-1) gibberellic acid, 30 g l(-1) sucrose and 2.5 g l(-1) gellan gum. Embryo rescue as ovary culture with immature seeds 12(th)-16(th) day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents.

14.
Methods Mol Biol ; 877: 233-45, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22610632

RESUMO

In breeding of ornamental plants, interspecific hybridization and polyploidization have successfully been used to produce novel cultivars with blended traits of both parents and to introgress useful traits of one species to another. Embryo rescue techniques and molecular cytogenetic methods have successfully been used to produce and characterize interspecific hybrids in various genera. In this paper, recent advances in interspecific hybridization are described based on the results obtained in Primula, Cosmos, and Kalanchoe with special references to the use of embryo culture techniques for rescuing the abortive hybrid embryos. The methods for production and characterization of interspecific hybrids are categorized into three steps, i.e., (1) pollination, (2) rescue culture of immature embryo, and (3) confirmation of hybridity and ploidy level of the plants obtained. For interspecific crosses, emasculation step is usually needed to avoid self-pollination even in the genera with self-incompatibility system, such as Primula and Cosmos, since self-incompatibility is not always complete. Since interspecific crosses are usually hindered by various cross-incompatibility mechanisms, successful production of interspecific hybrids could be achieved only from limited crosses among those using many cultivars/strains of both parents, suggesting the importance of the selection of the compatible genotypes. Unilateral cross incompatibility is commonly observed in interspecific cross combinations, so reciprocal crosses should be conducted as an indispensable step. At the rescue culture step, addition of plant hormones, e.g., auxin cytokinin and gibberellin, to the culture medium at the appropriate concentrations is proved to be effective and necessary. The hybridity of the plants is efficiently confirmed at the seedling stage by DNA analysis in addition to the observation of morphological characters. The analysis of relative DNA contents by flow cytometry is an easy and rapid means to confirm hybridity and to estimate ploidy level and genomic combination.


Assuntos
Técnicas de Cultura Embrionária , Kalanchoe/embriologia , Primula/embriologia , Citometria de Fluxo , Genótipo , Kalanchoe/genética , Kalanchoe/crescimento & desenvolvimento , Primula/genética , Primula/crescimento & desenvolvimento
15.
Plant Cell Rep ; 30(6): 1041-53, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21293863

RESUMO

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).


Assuntos
Imunidade Inata/imunologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/imunologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Recombinação Genética/genética , Alquil e Aril Transferases/genética , Southern Blotting , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Marcadores Genéticos , Glucuronidase/metabolismo , Imunidade Inata/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Brotos de Planta/anatomia & histologia , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regeneração
16.
Biotechnol Lett ; 33(6): 1249-55, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21287228

RESUMO

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.


Assuntos
Cucumovirus/genética , Cucumovirus/patogenicidade , Genes Virais , Lilium/genética , Lilium/virologia , Agrobacterium tumefaciens/genética , Sequência de Bases , Cucumovirus/enzimologia , DNA Viral/genética , Vírus Defeituosos/enzimologia , Vírus Defeituosos/genética , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas , RNA Replicase/genética , Transformação Genética
17.
Plant Cell Rep ; 30(4): 587-97, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21184230

RESUMO

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.


Assuntos
Alquil e Aril Transferases/fisiologia , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/enzimologia , Solanum tuberosum/enzimologia , Agrobacterium tumefaciens/genética , Alquil e Aril Transferases/genética , Alternaria/patogenicidade , Northern Blotting , Southern Blotting , Western Blotting , Fusarium/patogenicidade , Imunidade Inata/genética , Imunidade Inata/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Reação em Cadeia da Polimerase , Solanum tuberosum/genética , Solanum tuberosum/microbiologia
18.
Plant Cell Rep ; 29(9): 943-54, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20552202

RESUMO

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.


Assuntos
Alternaria/patogenicidade , Cucurbitaceae/genética , Defensinas/genética , Fusarium/patogenicidade , Imunidade Inata , Agrobacterium tumefaciens/genética , Cucurbitaceae/imunologia , DNA de Plantas/genética , Defensinas/imunologia , Doenças das Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Técnicas de Cultura de Tecidos , Transformação Genética , Transgenes
19.
Methods Mol Biol ; 589: 127-39, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20099097

RESUMO

Numerous shoots were directly regenerated from the leaf explants of Lysionotus pauciflorus on the MS medium containing 0.5-2 microM NAA with or without 1 microM BA. The calli were induced from the leaves on MS medium supplemented with 2 microM 2, 4-D. The calli proliferated about four times in fresh weight in the liquid medium of the same composition as the callus induction medium after 4 weeks of culture on a rotary shaker at 100 rpm. Shoots were induced from these calli on the regeneration medium amended with 32 microM BA or 0.5 microM zeatin. Regenerated shoots rooted easily on (1/2) MS medium without any plant growth regulators. Most of the regenerants from callus were diploid, whereas eight of 66 acclimatized plantlets were tetraploid determined by flow cytometric analysis. Furthermore, flow cytometric analysis of calli also revealed tetraploidy.


Assuntos
Técnicas de Cultura , Magnoliopsida/crescimento & desenvolvimento , Aclimatação , Proliferação de Células , Técnicas de Cultura/instrumentação , Citometria de Fluxo , Magnoliopsida/efeitos dos fármacos , Magnoliopsida/genética , Reguladores de Crescimento de Planta/farmacologia , Folhas de Planta/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Ploidias , Regeneração , Fatores de Tempo
20.
Plant Cell Rep ; 26(6): 735-43, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17205333

RESUMO

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.


Assuntos
Orchidaceae/fisiologia , Rhizobium/fisiologia , Transformação Bacteriana , Sequência de Bases , Cinamatos , Meios de Cultura , Primers do DNA , Higromicina B/análogos & derivados , Plantas Geneticamente Modificadas , Sacarose
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