The objectives of this study are: (i): To investigate and recognize the internal and abnormalities impacts induced by phytoplasma infection in the tomato host according to recent studies have shown that the association between plants and phytoplasmas can result in anatomical alteration in phloem tissues of infected plants, and great differences between healthy and diseased samples using microscopic examination of longitudinal, cross or ultra-thin sections of leaf blade, leaf petiole and stem. (ii): To determine the efficiency of different techniques toward production of phytoplasma-free tomato plantlets and mitigation of phytoplasma disease.
CHITINASE AS THE MOST IMPORTANT SECONDARY METABOLITES OF STREPTOMYCES BACTERISIJSIT Editor
Fungal phytopathogens pose serious problems worldwide in the cultivation of economi cally
important plants.
Chemical fungicides are extensively used in current agriculture.However, excessive use of chemical
fungicides in agriculture has led to deteriorating human health , environmental pollution, damaged to
ecosystem and development of pathogen resistance to fungicide.
Because of the worsening problems in fungal disease control , a serious search is needed to identify
alternative methods for plant protection, which are less dependent on chemicals and are more
environmentally friendly. Microbial antagonists are widely used for the biocontrol of fungal plant diseases.
Many species of actinomycates, particulary those belonging to the genus sterptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi.
Another way biological control has been developed as an alternative of chemicals to tock with plant
pathogenic fungi. Considering high presence of chitin in fungal cell wall, chitinase enzyme is camped as an
effective biocontrol agent against phytopathogenic fungi. Streptomyces bacteria are able to produce various chitinase enzymes, chitinases produced by streptomyces belong to the families 18 and 19 glycosyl hydrolases.
The antifungal activity is mostly shown by fomily 19 Chitinases. In comparison with bacterial family 18
chitinases, the specific hydrolyzing activity of chitinase 19 against soluble and in soluble chitinous substrates
has been markedly higher. Considering the importance of family to investigate antifungal potential of
streptomyces bacteria isolated from east Azarbijan region soils based on molecular identification of family 19
chitinase. encoding gene in these bacteria.
To aim the purpose 110 soil samples were collected from East Azarbaijan and 310 strepomyces
isolates were selected using macroscopic and microscopic observations. DNA genomic of all of the isolates
were extracted and PCR reactions was done using chitinase 19 designed primers as marker.
Totally isolates were selected with molecular selection and antagonistic test were done. One of the isolates
exhibit the most strong antifungal activity.
The strain was identified using 16srDNA gene, and the chitinase encoding gene were amplified partially to
prove the PCR selection. Finally the bacterium were introduced as potentially biological fertilizer.
Prevalence, occurrence and biochemical characterization of Xanthomonas campes...INNS PUBNET
Xanthomonas campestris pv. vesicatoria the causal organism of bacterial spot in tomato results in heavy losses both in the form of quality and. In this study a survey was carried out to report the incidence of bacterial spot disease of tomato in district Swat. We reported maximum disease incidence in tehsil Kabal (71.66%), followed by Charbagh (61.66%) and Barikot (58.33%). For resistant screening a total of 13 tomato germplasms were screened against the disease. The foliar severity ranged from 3.33% to 73.33%, while severity for fruits was ranged from 18.33% to 30.66%. In case of phenotypic data the highest numbers of fruits obtained were 34, plant height 79.5cm and fruit weight was 470 grams/ten tomatoes. While the lowest average numbers of fruits were 6.67, plant height 45.7cm and fruit weight recorded was 215.67 grams/ten tomatoes. Line 1288 showed highest level of resistance followed by Red-stone. However, line 9708 showed highest susceptibility when exposed to artificial inoculation. Our study showed that bacterial spot is a major issue in some part of Pakistan and germplasm screening are linked to increased host resistance and could offer an important contribution to future integrated bacterial spot management programs.
MOLECULAR ANALYSIS OF BACTERIAL GENE CODING CHITINASE ENZYMES, FAMILY 19 STR...IJSIT Editor
Fungal phytopathogens pose serious problems worldwide in the cultivation of economically
important plants.
Chemical fungicides are extensively used in current agriculture.However, excessive use of chemical
fungicides in agriculture has led to deteriorating human health , environmental pollution, damaged to
ecosystem and development of pathogen resistance to fungicide.
Because of the worsening problems in fungal disease control , a serious search is needed to identify
alternative methods for plant protection, which are less dependent on chemicals and are more
environmentally friendly. Microbial antagonists are widely used for the biocontrol of fungal plant diseases.
Many species of actinomycates, particulary those belonging to the genus sterptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi.
Another way biological control has been developed as an alternative of chemicals to tock with plant
pathogenic fungi. Considering high presence of chitin in fungal cell wall, chitinase enzyme is camped as an
effective biocontrol agent against phytopathogenic fungi. Streptomyces bacteria are able to produce various chitinase enzymes, chitinases produced by streptomyces belong to the families 18 and 19 glycosyl hydrolases.
The antifungal activity is mostly shown by fomily 19 Chitinases. In comparison with bacterial family 18
chitinases, the specific hydrolyzing activity of chitinase 19 against soluble and in soluble chitinous substrates
has been markedly higher. Considering the importance of family to investigate antifungal potential of
streptomyces bacteria isolated from east Azarbijan region soils based on molecular identification of family 19
chitinase. encoding gene in these bacteria.
To aim the purpose 110 soil samples were collected from East Azarbaijan and 310 strepomyces
isolates were selected using macroscopic and microscopic observations. DNA genomic of all of the isolates
were extracted and PCR reactions was done using chitinase 19 designed primers as marker.
Totally isolates were selected with molecular selection and antagonistic test were done. One of the isolates
exhibit the most strong antifungal activity.
The strain was identified using 16srDNA gene, and the chitinase encoding gene were amplified partially to
prove the PCR selection. Finally the bacterium were introduced as potentially biological fertilizer.
Identification and Evaluation of Antifungal Compounds from Botanicals for th...researchagriculture
Red rot is a devastating disease in sugarcane caused by fungus,
Colletotrichum
falcatum
. In this study, eighteen different botanicals were screened for
identifying effective antifungal compound against
C.
falcatum.
Among the plants
screened, 15 per cent aqueous leaf extract of
Psoralea corylifolia
alone inhibited 100
per cent growth of both mycelium as well as spore germination under
in vitro
conditions. The extract did not exhibit any inhibitory effect to the beneficial microbes
viz.
,
Pseudomonas fluorescens
,
Bacillus megaterium
and
Gluconacetobacter
diazotrophicus
which are normally used in sugarcane. The effective plant extracts
exhibiting 100 per cent antifungal activity was subjected to TLC, HPLC and GC
-
MS
analysis to identify the bioactive antifungal compound. It revealed the
presence of
7H
-
furo [3,2
-
G] (1) benzopyran
-
7
-
one as main bioactive compound which is thought to be
the intermediate of antifungal compound, 8
–
methoxypsoralen formed during
biosynthesis.
Biotechnology in capsule form. Plant biotechnology . Industry related information. Latest info. Different aspects of plant biotechnology. Cell biology. industrial biotechnology.
CHITINASE AS THE MOST IMPORTANT SECONDARY METABOLITES OF STREPTOMYCES BACTERISIJSIT Editor
Fungal phytopathogens pose serious problems worldwide in the cultivation of economi cally
important plants.
Chemical fungicides are extensively used in current agriculture.However, excessive use of chemical
fungicides in agriculture has led to deteriorating human health , environmental pollution, damaged to
ecosystem and development of pathogen resistance to fungicide.
Because of the worsening problems in fungal disease control , a serious search is needed to identify
alternative methods for plant protection, which are less dependent on chemicals and are more
environmentally friendly. Microbial antagonists are widely used for the biocontrol of fungal plant diseases.
Many species of actinomycates, particulary those belonging to the genus sterptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi.
Another way biological control has been developed as an alternative of chemicals to tock with plant
pathogenic fungi. Considering high presence of chitin in fungal cell wall, chitinase enzyme is camped as an
effective biocontrol agent against phytopathogenic fungi. Streptomyces bacteria are able to produce various chitinase enzymes, chitinases produced by streptomyces belong to the families 18 and 19 glycosyl hydrolases.
The antifungal activity is mostly shown by fomily 19 Chitinases. In comparison with bacterial family 18
chitinases, the specific hydrolyzing activity of chitinase 19 against soluble and in soluble chitinous substrates
has been markedly higher. Considering the importance of family to investigate antifungal potential of
streptomyces bacteria isolated from east Azarbijan region soils based on molecular identification of family 19
chitinase. encoding gene in these bacteria.
To aim the purpose 110 soil samples were collected from East Azarbaijan and 310 strepomyces
isolates were selected using macroscopic and microscopic observations. DNA genomic of all of the isolates
were extracted and PCR reactions was done using chitinase 19 designed primers as marker.
Totally isolates were selected with molecular selection and antagonistic test were done. One of the isolates
exhibit the most strong antifungal activity.
The strain was identified using 16srDNA gene, and the chitinase encoding gene were amplified partially to
prove the PCR selection. Finally the bacterium were introduced as potentially biological fertilizer.
Prevalence, occurrence and biochemical characterization of Xanthomonas campes...INNS PUBNET
Xanthomonas campestris pv. vesicatoria the causal organism of bacterial spot in tomato results in heavy losses both in the form of quality and. In this study a survey was carried out to report the incidence of bacterial spot disease of tomato in district Swat. We reported maximum disease incidence in tehsil Kabal (71.66%), followed by Charbagh (61.66%) and Barikot (58.33%). For resistant screening a total of 13 tomato germplasms were screened against the disease. The foliar severity ranged from 3.33% to 73.33%, while severity for fruits was ranged from 18.33% to 30.66%. In case of phenotypic data the highest numbers of fruits obtained were 34, plant height 79.5cm and fruit weight was 470 grams/ten tomatoes. While the lowest average numbers of fruits were 6.67, plant height 45.7cm and fruit weight recorded was 215.67 grams/ten tomatoes. Line 1288 showed highest level of resistance followed by Red-stone. However, line 9708 showed highest susceptibility when exposed to artificial inoculation. Our study showed that bacterial spot is a major issue in some part of Pakistan and germplasm screening are linked to increased host resistance and could offer an important contribution to future integrated bacterial spot management programs.
MOLECULAR ANALYSIS OF BACTERIAL GENE CODING CHITINASE ENZYMES, FAMILY 19 STR...IJSIT Editor
Fungal phytopathogens pose serious problems worldwide in the cultivation of economically
important plants.
Chemical fungicides are extensively used in current agriculture.However, excessive use of chemical
fungicides in agriculture has led to deteriorating human health , environmental pollution, damaged to
ecosystem and development of pathogen resistance to fungicide.
Because of the worsening problems in fungal disease control , a serious search is needed to identify
alternative methods for plant protection, which are less dependent on chemicals and are more
environmentally friendly. Microbial antagonists are widely used for the biocontrol of fungal plant diseases.
Many species of actinomycates, particulary those belonging to the genus sterptomyces, are well known as
antifungal biocontrol agents that inhibit several plant pathogenic fungi.
Another way biological control has been developed as an alternative of chemicals to tock with plant
pathogenic fungi. Considering high presence of chitin in fungal cell wall, chitinase enzyme is camped as an
effective biocontrol agent against phytopathogenic fungi. Streptomyces bacteria are able to produce various chitinase enzymes, chitinases produced by streptomyces belong to the families 18 and 19 glycosyl hydrolases.
The antifungal activity is mostly shown by fomily 19 Chitinases. In comparison with bacterial family 18
chitinases, the specific hydrolyzing activity of chitinase 19 against soluble and in soluble chitinous substrates
has been markedly higher. Considering the importance of family to investigate antifungal potential of
streptomyces bacteria isolated from east Azarbijan region soils based on molecular identification of family 19
chitinase. encoding gene in these bacteria.
To aim the purpose 110 soil samples were collected from East Azarbaijan and 310 strepomyces
isolates were selected using macroscopic and microscopic observations. DNA genomic of all of the isolates
were extracted and PCR reactions was done using chitinase 19 designed primers as marker.
Totally isolates were selected with molecular selection and antagonistic test were done. One of the isolates
exhibit the most strong antifungal activity.
The strain was identified using 16srDNA gene, and the chitinase encoding gene were amplified partially to
prove the PCR selection. Finally the bacterium were introduced as potentially biological fertilizer.
Identification and Evaluation of Antifungal Compounds from Botanicals for th...researchagriculture
Red rot is a devastating disease in sugarcane caused by fungus,
Colletotrichum
falcatum
. In this study, eighteen different botanicals were screened for
identifying effective antifungal compound against
C.
falcatum.
Among the plants
screened, 15 per cent aqueous leaf extract of
Psoralea corylifolia
alone inhibited 100
per cent growth of both mycelium as well as spore germination under
in vitro
conditions. The extract did not exhibit any inhibitory effect to the beneficial microbes
viz.
,
Pseudomonas fluorescens
,
Bacillus megaterium
and
Gluconacetobacter
diazotrophicus
which are normally used in sugarcane. The effective plant extracts
exhibiting 100 per cent antifungal activity was subjected to TLC, HPLC and GC
-
MS
analysis to identify the bioactive antifungal compound. It revealed the
presence of
7H
-
furo [3,2
-
G] (1) benzopyran
-
7
-
one as main bioactive compound which is thought to be
the intermediate of antifungal compound, 8
–
methoxypsoralen formed during
biosynthesis.
Biotechnology in capsule form. Plant biotechnology . Industry related information. Latest info. Different aspects of plant biotechnology. Cell biology. industrial biotechnology.
— The microbiological content of Lettuce (a vegetable), commonly vended in the Benin metropolis of Edo state were evaluated. Five vending locations were chosen for the study. Whole and soft rot samples were purchased and analysed for microbiological composition. Results showed high counts in soft rot samples in lettuce. Nutrient agar plated lettuce samples had bacterial counts in the range of 2.0x 103 to 4.7x10 7. Pseudomonas species was the dominant species found in lettuce samples. Bacillus species was isolated from one location in the lettuce samples. Mac Conkey agar plated lettuce plated had bacterial counts in the range of 2.3 x 10 3 to 5.7x 10 7. Enterobacter species, E. coli, and Klebsiella species were the dominant species isolated. Though, Proteus species was isolated from lettuce samples obtained from location five only. The study observes that consuming soft rot samples could pose a risk of introducing pathogens to the consumer due to their high microbial counts and could be detrimental to the health of the consumer.
I have discussed Applications of Plant Tissue Culture under the following subheadings,
1. Micro Propagation
2. Clonal Propagation
3. Production of Genetically Variable Plants
4. Production of Virus Free Plants
5. Plant Breeding
6. Production of Useful Biochemicals
7. Preservation of Plant Genetic Resources
8. Importance of Tissue Culture in Biotechnology
1. Introduction: Tissue Culture is the in vitro culture of cells, tissues, organs or whole plant under controlled nutritional and environmental Conditions(T. Thorp, 2007).
The science of plant tissue culture takes its roots from the discovery of Cells (Robert Hooke in 1665) and propounding of cell theory.
In 1838, Schleiden and Schwann proposed that cell is the basic structural unit of all living organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plants.
2. Plant Tissue Culture: Past & Present Prospects
In 1902, a German physiologist, Gottieb Haberlandt for the first time attempted to culture isolated single palisade cells from leaves in knop’s salt solution.
The cell remained alive for up to 1 month, increased in size, accumulated starch but failed to divide.
Though he was unsuccessful but he laid the foundation of tissue culture so he is regarded as Father of Plant Tissue Culture.
In the Subsequent years different landmark discoveries were made. Some of them are:
Use of specialized media for aseptic culture of Orchid seeds (Knudson, 1925) and other workers also demonstrated that plants could be propagated in vitro from the minuscule seeds of the Orchidaceae.
Further culture of other plant tissue was not possible due to lack of knowledge of the specific hormones to be added to the culture media.
This limitation was overcomed by the elucidation of the nature of Auxin, IAA, by Thimann and Went(1930) that plants would be subsequently regenerated through the use of IAA or its analogs.
Discovery of Cytokinins, specially Kinetin(6-furfurylaminopurine) by Miller et al. (1956), the regeneration of intact plants from tissue of many herbaceous species became a practical reality.
Effect of interaction between different plant growth regulators on in vitro s...Agriculture Journal IJOEAR
— In this paper a shoot multiplication is described for Citrus latifolia Tan. (persian lime) using nodal segment explants of young one – old – year trees by two different pathways contain with and without callusing phase. The best result for multiple shoot formation and regenerated shoot formation was 3.2 and 2.6 shoots per explants with 4.44 µM BA plus 0.053 µM NAA and 4.44 µM BA plus 0.049 µM IBA respectively. Alike shoot regeneration, shoot elongation was occurred in medium with 4.44 µM BA and 0.049 µM IBA. Micropropagated and regenerated plants are under other experiments. Abbreviation: BA – 6 benzylaminopurine; IBA – Indole acetic acid; NAA – Naphtalene acetic acid; PGRs – Plant Growth Regulators.
Somaclonal Variation: A new dimension for sugarcane improvementDr. siddhant
Plant tissue culture or micropropagation technique is the rapid method to multiply newly released cultivar in limited
time. Crop improvement by conventional method in vegetatively propagated crops like sugarcane is very difficult due to
its narrow genetic base and other limitations. Somaclonal variations are easily achieved in asexually propagated crops
like sugarcane and banana. Tissue culture derived variations are known as somaclonal variation. These variations play
an important role in crop improvement program. Genetic variations are heritable in next generation and important for
crop improvement, epigenetic changes are temporary ultimately reversible. Mutation breeding is also very advantageous
for improving a cultivar. Somaclonal variants of sugarcane are available for several traits like drought, salt tolerance, red
rot, eye spot disease, quality and quantity trait. Molecular marker techniques like RFLP, RAPD, AFLP and SSR etc. are
regularly used preferentially over traditional phenotypic or cytological methods.
Unit 2 plant tissue culture applications, advantages and limitationsDr. Mafatlal Kher
This presentation is related to the application of plant tissue culture techniques in various sectors, and it also highlights the advantages and limitations of plant tissue culture
Isolation, identification of antagonistic rhizobacterial strains obtained fro...Shazia Shahzaman
Plant growth promoting rhizobacteria (PGPR), are associated with roots, found in the rhizosphere and can directly or indirectly enhance the plant growth. In this study soil was collected from rhizosphere of chickpea fields of different areas of Rawalpindi division of Pakistan. PGPR were isolated, screened and characterized. Eight isolates of rhizobacteria (RHA, RPG, RFJ, RC, RTR, RT and RK) were isolated from Rawalpindi division and were characterized. The antagonistic activity of these PGPR isolates against root infecting fungi (Fusarium oxysporum and Verticillium spp.,) was done and production of indole acetic acid (IAA), siderophore and P-solubilization was evaluated. The isolates RHA, RPG, RFJ, RC, RRD and RT were found to be positive in producing siderophore, IAA and P-solubilization. Furthermore, most of the isolates showed antifungal activity against Fusarium oxysporum, and Verticillium spp. The rhizobacterial isolates RHA, RPG, RFJ, RC, RRD, RTR, RT and RK were used as bio-inoculants that might be beneficial for chickpea cultivation as the rhizobacterial isolates possessed the plant growth promoting characters i.e. siderophore, IAA production, phosphate solubilization. In in vitro tests, Pseudomonas sp. and Bacillus spp. inhibited the mycelial growth of the fungal root pathogens. The isolates (RHA and RPG) also significantly increased (60-70%) seed germination, shoot length, root length of the chickpea. The incidence of fungi was reduced by the colonization of RHA and RPG which enhanced the seedling vigor index and seed germination. The observations revealed that isolates RHA and RPG is quite effective to reduce the fungal root infection in greenhouse, and also increases seed yields significantly. These rhizobacterial isolates appear to be efficient yield increasing as well as effective biocontrol agent against fungal root pathogen.
Applications of Plant Tissue Culture || Presented by Mamoona Ghaffar Mamoona Ghaffar
Applications of Plant Tissue Culture || Presented by Mamoona Ghaffar
Applications in Genetic Engineering, Transgenic Plants, Biotechnology, Industries
Feel free to ask about your queries.
The objectives of this study to investigate the occurrence and etiology of tomato malformation recently observed in Egypt and illustrate the responsibility of associated phytoplasma. Also, the study was extended to characterize associated phytoplasma, based on molecular techniques.
Effect of Clariodeoglomusclariodeorum on morphology and abundant of carrot ro...AI Publications
The roothairs are important components of the root for absorbing nutrients for plants and also secreting the plant-produced secretes. Morphology and their number are influenced by various environmental and internal factors and are regulated by them. Mycorrhizal fungi are established through the root and their presence in the root can affect root’s physical and chemical properties.The aim of this study was to evaluate the effect of the presence of the fungus in the roots on the characteristics of the capillary roots.In order to remove the effect of other microorganisms on the results of the experiment, this experiment was performed in vitro on the roots of the carrot secondary phloem tissue culture. In this experiment, the fungus could affect the characteristics of the root hairs: their number (22% decrease in the root hairs number in the root hair area of the root) and their length (A decrease of 21.3% in the length of capillary roots in mycorrhizal plants). These changes in the characteristics of capillary roots were also caused by the presence of fungal structures in the roots as well as by the decrease in the production of strigolactones. In this study, changes in the production of strigolactones calculated by using their effect on seed germination of Phelipancheaegyptiaca.
— The microbiological content of Lettuce (a vegetable), commonly vended in the Benin metropolis of Edo state were evaluated. Five vending locations were chosen for the study. Whole and soft rot samples were purchased and analysed for microbiological composition. Results showed high counts in soft rot samples in lettuce. Nutrient agar plated lettuce samples had bacterial counts in the range of 2.0x 103 to 4.7x10 7. Pseudomonas species was the dominant species found in lettuce samples. Bacillus species was isolated from one location in the lettuce samples. Mac Conkey agar plated lettuce plated had bacterial counts in the range of 2.3 x 10 3 to 5.7x 10 7. Enterobacter species, E. coli, and Klebsiella species were the dominant species isolated. Though, Proteus species was isolated from lettuce samples obtained from location five only. The study observes that consuming soft rot samples could pose a risk of introducing pathogens to the consumer due to their high microbial counts and could be detrimental to the health of the consumer.
I have discussed Applications of Plant Tissue Culture under the following subheadings,
1. Micro Propagation
2. Clonal Propagation
3. Production of Genetically Variable Plants
4. Production of Virus Free Plants
5. Plant Breeding
6. Production of Useful Biochemicals
7. Preservation of Plant Genetic Resources
8. Importance of Tissue Culture in Biotechnology
1. Introduction: Tissue Culture is the in vitro culture of cells, tissues, organs or whole plant under controlled nutritional and environmental Conditions(T. Thorp, 2007).
The science of plant tissue culture takes its roots from the discovery of Cells (Robert Hooke in 1665) and propounding of cell theory.
In 1838, Schleiden and Schwann proposed that cell is the basic structural unit of all living organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plants.
2. Plant Tissue Culture: Past & Present Prospects
In 1902, a German physiologist, Gottieb Haberlandt for the first time attempted to culture isolated single palisade cells from leaves in knop’s salt solution.
The cell remained alive for up to 1 month, increased in size, accumulated starch but failed to divide.
Though he was unsuccessful but he laid the foundation of tissue culture so he is regarded as Father of Plant Tissue Culture.
In the Subsequent years different landmark discoveries were made. Some of them are:
Use of specialized media for aseptic culture of Orchid seeds (Knudson, 1925) and other workers also demonstrated that plants could be propagated in vitro from the minuscule seeds of the Orchidaceae.
Further culture of other plant tissue was not possible due to lack of knowledge of the specific hormones to be added to the culture media.
This limitation was overcomed by the elucidation of the nature of Auxin, IAA, by Thimann and Went(1930) that plants would be subsequently regenerated through the use of IAA or its analogs.
Discovery of Cytokinins, specially Kinetin(6-furfurylaminopurine) by Miller et al. (1956), the regeneration of intact plants from tissue of many herbaceous species became a practical reality.
Effect of interaction between different plant growth regulators on in vitro s...Agriculture Journal IJOEAR
— In this paper a shoot multiplication is described for Citrus latifolia Tan. (persian lime) using nodal segment explants of young one – old – year trees by two different pathways contain with and without callusing phase. The best result for multiple shoot formation and regenerated shoot formation was 3.2 and 2.6 shoots per explants with 4.44 µM BA plus 0.053 µM NAA and 4.44 µM BA plus 0.049 µM IBA respectively. Alike shoot regeneration, shoot elongation was occurred in medium with 4.44 µM BA and 0.049 µM IBA. Micropropagated and regenerated plants are under other experiments. Abbreviation: BA – 6 benzylaminopurine; IBA – Indole acetic acid; NAA – Naphtalene acetic acid; PGRs – Plant Growth Regulators.
Somaclonal Variation: A new dimension for sugarcane improvementDr. siddhant
Plant tissue culture or micropropagation technique is the rapid method to multiply newly released cultivar in limited
time. Crop improvement by conventional method in vegetatively propagated crops like sugarcane is very difficult due to
its narrow genetic base and other limitations. Somaclonal variations are easily achieved in asexually propagated crops
like sugarcane and banana. Tissue culture derived variations are known as somaclonal variation. These variations play
an important role in crop improvement program. Genetic variations are heritable in next generation and important for
crop improvement, epigenetic changes are temporary ultimately reversible. Mutation breeding is also very advantageous
for improving a cultivar. Somaclonal variants of sugarcane are available for several traits like drought, salt tolerance, red
rot, eye spot disease, quality and quantity trait. Molecular marker techniques like RFLP, RAPD, AFLP and SSR etc. are
regularly used preferentially over traditional phenotypic or cytological methods.
Unit 2 plant tissue culture applications, advantages and limitationsDr. Mafatlal Kher
This presentation is related to the application of plant tissue culture techniques in various sectors, and it also highlights the advantages and limitations of plant tissue culture
Isolation, identification of antagonistic rhizobacterial strains obtained fro...Shazia Shahzaman
Plant growth promoting rhizobacteria (PGPR), are associated with roots, found in the rhizosphere and can directly or indirectly enhance the plant growth. In this study soil was collected from rhizosphere of chickpea fields of different areas of Rawalpindi division of Pakistan. PGPR were isolated, screened and characterized. Eight isolates of rhizobacteria (RHA, RPG, RFJ, RC, RTR, RT and RK) were isolated from Rawalpindi division and were characterized. The antagonistic activity of these PGPR isolates against root infecting fungi (Fusarium oxysporum and Verticillium spp.,) was done and production of indole acetic acid (IAA), siderophore and P-solubilization was evaluated. The isolates RHA, RPG, RFJ, RC, RRD and RT were found to be positive in producing siderophore, IAA and P-solubilization. Furthermore, most of the isolates showed antifungal activity against Fusarium oxysporum, and Verticillium spp. The rhizobacterial isolates RHA, RPG, RFJ, RC, RRD, RTR, RT and RK were used as bio-inoculants that might be beneficial for chickpea cultivation as the rhizobacterial isolates possessed the plant growth promoting characters i.e. siderophore, IAA production, phosphate solubilization. In in vitro tests, Pseudomonas sp. and Bacillus spp. inhibited the mycelial growth of the fungal root pathogens. The isolates (RHA and RPG) also significantly increased (60-70%) seed germination, shoot length, root length of the chickpea. The incidence of fungi was reduced by the colonization of RHA and RPG which enhanced the seedling vigor index and seed germination. The observations revealed that isolates RHA and RPG is quite effective to reduce the fungal root infection in greenhouse, and also increases seed yields significantly. These rhizobacterial isolates appear to be efficient yield increasing as well as effective biocontrol agent against fungal root pathogen.
Applications of Plant Tissue Culture || Presented by Mamoona Ghaffar Mamoona Ghaffar
Applications of Plant Tissue Culture || Presented by Mamoona Ghaffar
Applications in Genetic Engineering, Transgenic Plants, Biotechnology, Industries
Feel free to ask about your queries.
The objectives of this study to investigate the occurrence and etiology of tomato malformation recently observed in Egypt and illustrate the responsibility of associated phytoplasma. Also, the study was extended to characterize associated phytoplasma, based on molecular techniques.
Effect of Clariodeoglomusclariodeorum on morphology and abundant of carrot ro...AI Publications
The roothairs are important components of the root for absorbing nutrients for plants and also secreting the plant-produced secretes. Morphology and their number are influenced by various environmental and internal factors and are regulated by them. Mycorrhizal fungi are established through the root and their presence in the root can affect root’s physical and chemical properties.The aim of this study was to evaluate the effect of the presence of the fungus in the roots on the characteristics of the capillary roots.In order to remove the effect of other microorganisms on the results of the experiment, this experiment was performed in vitro on the roots of the carrot secondary phloem tissue culture. In this experiment, the fungus could affect the characteristics of the root hairs: their number (22% decrease in the root hairs number in the root hair area of the root) and their length (A decrease of 21.3% in the length of capillary roots in mycorrhizal plants). These changes in the characteristics of capillary roots were also caused by the presence of fungal structures in the roots as well as by the decrease in the production of strigolactones. In this study, changes in the production of strigolactones calculated by using their effect on seed germination of Phelipancheaegyptiaca.
Potentials of Compost Tea of Certain Botanicals for Minimizing Root- Knot and...Premier Publishers
Two greenhouse experiments were conducted on eggplant to assess the impact of compost tea derived from plant residues namely rice hull (RHC), rice straw (RSC), tomato (TC), potato (PC), citrus (CC), and guava(GC) as well as city waste (CWC) compost on eggplant biomass and reproduction of Meloidogyne incognita and Rotylenchulus reniformis. Screened composts were introduced to plants as drenching application. Results indicated that percentage of increase in total plant fresh weight of eggplant infected with such nematodeswere more pronounced (P<0.05) with compost teas of RSW. Drenching the soil with RHC (Rf=0.7) and PC (Rf=0.9) were also effective in suppressing densities of M. incognita as well as number of galls, and eggs/ root. However, population densities and fecundity of R. reniformis were significantly reduced following the introduction of GC. Only, total phenol showed remarkable increment in plants treated with PC, GC and RSC compared to untreated inoculated plants.
Identification and evaluation of antifungal compounds from botanicals for the...researchagriculture
Red rot is a devastating disease in sugarcane caused by fungus, Colletotrichum falcatum. In this study, eighteen different botanicals were screened for identifying effective antifungal compound against C. falcatum. Among the plants screened, 15 per cent aqueous leaf extract of Psoralea corylifolia alone inhibited 100 per cent growth of both mycelium as well as spore germination under in vitro conditions. The extract did not exhibit any inhibitory effect to the beneficial microbes viz., Pseudomonas fluorescens, Bacillus megaterium and Gluconacetobacter diazotrophicus which are normally used in sugarcane. The effective plant extracts exhibiting 100 per cent antifungal activity was subjected to TLC, HPLC and GC-MS analysis to identify the bioactive antifungal compound. It revealed the presence of 7H-furo [3,2-G] (1) benzopyran-7-one as main bioactive compound which is thought to be the intermediate of antifungal compound, 8 – methoxypsoralen formed during biosynthesis.
Article Citation:
Rajkumar D and Murugesan R.
Identification and Evaluation of Antifungal Compounds from Botanicals for the Control of Sugarcane Red Rot Pathogen, Colletotrichum falcatum.
Journal of Research in Agriculture (2013) 2(1): 164-172.
Full Text:
http://www.jagri.info/documents/AG0044.pdf
Ecklonia Maxima Extract Effect in Tissue Regeneration of Symbionts at in Vivo...IJEAB
The research was conducted to determine the Ecklonia maxima extract effect in the symbiont accretion at the in vivo heteroplasmic grafting of some tomatoes. E. maxima or sea bamboo is a seaweed used for obtaining of organic extracts used as stimulators in horticulture because consists the natural plant hormones such as auxins and cytokinins which have optimal role in cell division, important activity for tissue regeneration. The experimental variants were grafted plants, combinations between different symbionts, cultivar fragments from Lycopersicon esculentum specie. The symbionts were two scions, F1 hybrids, 'Siriana' (Romanian cultivar), 'Abellus' (Dutch cultivar) and two rootstocks, 'Buzău' variety (Romanian cultivar), 'Emperador' F1 hybrid (Dutch cultivar). The algae extract used had auxins (11 mg/L) and cytokinins (0.3 mg/L). Two treatments were applied before grafting on scion and rootstock, 1 mL/500 mL water and a treatment at grafting on soil, 5 mL/L water. Control variant was without hormone extract. Determinations, observations and interpretations of the algae effect were made on symbionts. The best results on tissue regeneration were obtained in plants treated with sea bamboo extract compared to untreated control plants. The E. maxima extract influenced the tissue regeneration.
The objective of this study was to examine the antiviral activity of native lactoferrin against Potato virus x, the most important virus that severely affects potato crop and productivity in Egypt, using tissue culture technique and spraying the plants in greenhouse by the aqueous solution of lactoferrin.
Isolation of endophytes from potato and their antagonist effect against Fusar...Innspub Net
Plant endophytes may be intercellular or intracellular depending upon their location in the plant tissue because they are present inside the cells or in the intracellular space, respectively. Isolation of endophytic bacteria has been reported from both monocot and dicot plants, ranging from woody trees, such as teak and pear, to herbaceous crop plants such as mustard and maize. The aim of this study was the isolation of endophytes from potato and their antagonist effect against Fusarium oxysporum. Endophytic fungi were isolated from leaves, stems and roots of healthy Potato plant derived from Chak No.359/E.B Village, Tehsil Burewala. Isolation of endophytic fungi from plant parts was done according to the method described by Petrini. The media used in the present study was the Potatodextrose agar (PDA) for fungus and nutrient agar medium for maintaining bacterial stains. F.oxysporum was taken from the Plant pathology lab of UAF sub-campus Burewala-Vehari . The results of the experiment clearly revealed that the stems, root and leaf of the potato plants under present investigation had the maximum colonization frequency for fungal endophytes. Fusarium oxysporum showed rapid growth 5-7cm in5 days. Fusarium oxysporum was white and growing rapidly that later produced dark violet pigments in PDA. Erwinia showed light green, circular, shining, slimy, smooth characteristics. The isolate strain of Bacillus showed rodshaped, fuzzy white or slightly yellow circular and irregular characteristics.
The present study has been conducted to perform the following objectives:
1- Study the effect of different temperature degrees and meristem culture technique on elimination of Potato leafroll virus (PLRV) and Potato virus x (PVX), from the most commonly potato cultivars in Egypt (Spunta and Lady Rosette).
2- Production of potato minitubers from direct transplanting of in vitro virus-free plantlets in greenhouse. Also, investigate the effect of different soil mixtures on minitubers production for both cultivars.
Effect of Agrobacterium Induced Necrosis, Antibiotic Induced Phytotoxicity an...Sandip Magdum
Agrobacterium tumefaciens infection and antibiotic wash are the critical steps of Agrobacterium mediated plant transformation procedure, most time responsible for lower transformation efficiency due to necrosis and phytotoxicity caused by biotic stress of Agrobacterium and abiotic stress by antibiotics respectively. Ammi majus Egyptian origin medicinal plant and Pearl millet cereal grain crop were studied for their stress responses to Agrobacterium mediated transformation (AMT). Agrobacterium strains LBA4404 (O.D.=0.6-0.8) and EHA105 (O.D.=0.2-0.4) were used for transformation experiments to infect calli of Ammi majus and embryogenic calli of Pearl millet respectively. Incase of antibiotic wash, Cefotaxime 500 mg L-1 was used for LBA4404 infected Ammi majus calli and Timentin 300 mg L-1 was used for EHA105 infected embryogenic calli of Pearl millet.
Effects of Agrobacterium infection, antibiotic and NaOCl washes on Agrobacterium removal and both explants physiological changes during transformation experimental procedures were studied. At the end of the experiments explants survival efficiency of Ammi majus and pearl millet were 8% and 5% respectively. Biotic and abiotic stress factors responsible for lower efficiency were investigated with various other factors and strategies were discussed which are need to be considered for higher transformation events and target tissue survival.
Severity of Tomato brown rugose fruit virus in tomato (Solanum lycopersicum L...AI Publications
The purpose of this research was to describe the Tomato brown rugose fruit virus, from three isolates collected in the field and also to use a diagrammatic scale of severity for its evaluation. The isolation was carried out with the collection of 200 ha-1 leaflets, according to a statistical method, from commercial greenhouses. Agdia® immunological strips were used to confirm the symptoms and the virus was identified by RT-PCR. A completely randomized experimental design was established in tomato plants var. Río Grande®, with three treatments or isolates and five repetitions: Blindom F1® tissue, Quiroga® Enza zaiden tissue and Quiroga® Enza zaiden fruit; Controls with phosphate buffer and buffer+celite were used as negative control. The trial began with the inoculation of 45-day-old tomato plants, where only the primary leaves were inoculated. Fertilization was carried out twice a week using macro and microelements from commercial companies. To quantify the percentage of damage to foliage and fruit, a diagrammatic scale of severity was used. The three isolates evaluated differed in the symptoms produced by ToBRFV, where; The Fruto Quiroga® Enza zaiden isolate stood out, with a higher incidence, severity and shorter incubation period compared to the two isolates evaluated.
Early blight of potato caused by Alternaria alternata (Fries) Keissler is one of the factors that affect
potato production. Using chemical control to reduce disease severity represent another risk for
agriculture. Biological control, using microorganisms, as well as plant extract and compost are safe
measures that give reliable control. Applying either measure of control reduced disease incidence to less
than 2% during two successive seasons. Microbial spray of diseased potato reduced the disease to various
extents. Trichoderma sp. was the most effective among all tested microorganisms, followed by
Penicillium sp. On the other hand, alcoholic extract of three plants was more effective than their water
extract. However, water extract showed highly significant reduction of disease incidence. Polygonum
gave the highest reduction of disease incidence in both cases. Also water extract of compost greatly
suppressed early blight when applied as spray to potato leaves. Duration of extraction may affect the
ability of compost extract to suppress disease. All results were comparable to that of fungicides.
Effect of plant growth promoting rhizobacterial (PGPR) inoculation on growth ...IJEAB
Plant Growth promoting rhizobacteria are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with roots. They benefit plants through Production of plant hormones, such as auxins, asymbiotic N2 fixation, solubilization of mineral phosphates, antagonism against phytopathogenic microorganisms by production of antibiotics, siderophroes, Chitinase and other nutrients ability to effectively colonize roots are responsible for plant growth promotion. An experiment was conducted in the field of National Institute of Agronomic Research of Meknes. Morocco. The experiment was a completely randomized design with six replicates. There were four treatments viz. T1: (control; N0 -PGPR), T2: (N0 +2027-2), T3: (N0 +2066-7) and T4: (N0+2025-1). The results indicated that a remarkable increase in root growth, namely length, the diameter of the rod and the total chlorophyll. A total of three different bacteria colonies were isolated and proceed with in vitro screening for plant growth promoting activities; phosphate solubilization, nitrogen fixation, indole acetic acid (IAA), ammonia production and antimicrobial enzymes (cellulose, chitinase and protease) activity. Among the three bacterial strains, all bacterial strains are able to produce ammonia, IAA production and nitrogen fixation activity, one strain phosphate solubilizing activity, two strain are able to produce cellulase syntheses, Protease activity and Chitinase activity.
Evaluate the Efficiency of Gamma Irradiation and Chitosan on Shelf-Life of St...IJEABJ
Chitosan play an important role as an antifungal against Botrytis cinerea and the effect was a concentration dependent. The obtained results of in vitro experiment demonstrated that chitosan (4%) decreased radial growth of B. cinereato 2 %. Invivo the severity of infection reduced from 59.8 and 100.0 to 9.7, 33.8 and 40.1 in first, second and third week’s storage periods at 13C, respectively. Also, chitosan coating (4%) significantly caused an increase in fruit firmness whereas TSS was decreased with an increase by increasing in storage time. However,Vitamin C gave fluctuated results by increasing storage time. Gamma irradiation at 2.5 KGy reduced severity (%) of infected fruits from 55.5, 100 and 100 to 31.7, 45.9 and 49.9 and in healthy fruits severity (%) reduced from 48.9, 100 and 100 to 23.3, 25.1 and 29.1 in different storage periods 1, 2 and 3 weeks, respectively. Similarly, chitosan as well as gamma irradiation combination induced a significant increase of peroxidase enzyme (POD) activity. Induced changes in surface morphology and damage of cell structure caused by using chitosan shown by scanning electron microscopy. Also, gamma irradiation causes changes in hyphea structure and in surface morphology but combination of gamma irradiation with chitosan was more effective in altering fungus morphology and cell structure damage and no spore forming. This providing the efficiency of combination on reducing disease severity (%) of strawberry.
Similar to Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture techniques (20)
The First Workshop of Plant Pathology Research Institute
The Modern Trends in Controlling Plant Diseases
10-11 February 2019
Under auspices of
Prof. Dr. Eszzaldin Omar Abusteit
Minster of Agricultural and Land Reclamation
Prof. Dr. Mohamed Soliman
Director of Agricultural Research Center
&
President of the workshop
Prof. Dr. Ashraf El Saied Khalil
Director of Plant Pathology Research Institute
This work aimed to (i) Identify and characterize Onion yellow dwarf virus potyvirus (OYDV) in the onion plants in Egypt. (ii) Clone and sequence the coat protein gene of the Egyptian isolate of OYDV and comparing it with other OYDV isolates reported in the GenBank database. (iii) Study the influence of therapeutic doses of kinetin (6-Furfurylaminopurine) on production of virus-free onion plantlets and improve its regeneration ability through in vitro micropropagation.
Research topic was come from successful inactivation of some plant viruses by gamma irradiation like Citrus tristeza virus, Necrotic ring spot virus and Prune dwarf virus. Gamma irradiation has been also used to sterilize agricultural products in order to increase their conservation time or to reduce pathogen when being traded from a country to another. Gamma radiation is high-energy radiation emitted from certain radioactive isotopes as cobalt 60, these isotopes are potential sources of gamma radiation. Therefore, this research was conducted to find out the inactivation possibility of Hibiscus witches' broom (HibWB)-phytoplasma using gamma irradiation through tissue culture technique with clarify their effect on in vitro growth and survival rate.
The objectives of this study were to detect and characterize the phytoplasma in tissues of diseased hibiscus plants using Dains’ stain light microscopy and molecular based techniques. Molecular characterization was performed using the DNA sequencing and phylogenetic analysis of the spacer region between 16S and 23S rRNA fragment of the isolated phytoplasma genome. This work concerning phytoplasma associated witches' broom (group 16SrII) diseases of hibiscus plants is achieved for the first time in Egypt.
In Egypt isolates of Prunus necrotic ringspot virus (PNRSV) were detected in peach and apricot grooves (Abdel-Salam et al. 2008a), Rosa spp. (Abdel-Salam et al., 2008b) as well as on sugarbeet plantations (Abdel-Salam et al., 2006a).
In the present study, incidence of PNRSV is reported on apple (Malus domestica). Severe symptoms mimic infections with PNRSV were recently detected on apple from several orchards in the vicinity of Nubaria city, Beheira governorate. Infected-apple samples were brought to the laboratory for further detection at serological and molecular levels to check the presence of virus. The present study reports the presence of an isolate of PNRSV on apple, viz. PNRSV-Apple.
The present study aims to (I) evaluate the antiviral activity of eugenol oil nanoemulsion (EON) on eliminate Banana bunchy top virus (BBTV) from naturally infected banana plants and produce virus-free banana plants, (II) identify fungal contaminants of in vitro banana cultures and (III) evaluate the potential of EON on the suppression of the identified microbial contaminants and reduce of their occurrence frequency.
The initial objective of this work was to isolate Tomato spotted wilt virus (TSWV) from asymptomatic infected plants and then identify the virus on the basis of biological properties among the most common or other hosts if possible after inoculation. Phylogenetic analysis was then conducted to gain information about the similar identity of the TSWV-isolate that reported in this study with available TSWV sequences from other parts of the world.
زراعة الأنسجة النباتية
طرق الإكثار بزراعة الأنسجة النباتية
البيئة الغذائية المستخدمة فى الزراعة
مراحل زراعة الأنسجة النباتية
العوامل المؤثرة فى نجاح زراعة الأنسجة
العقبات التى تنشأ أثناء مراحل زراعة الأنسجة النباتية
مجالات زراعة الأنسجة النباتية
بدأت تقنية زراعة أنسجة النبات بهدف اكثار وإنتاج نباتات خالية من الأمراض النباتية، وتعددت أشكال هذه التقنية مثل زراعة القمم المرستيمية للنبات، وإستخدام العلاج الحرارى أو الكيماوي، وقد واكب هذا ظهور طرق حديثة فعالة تمثل مصادر جديدة للتغايرالوراثى فى برامج التربية وتحسين النبات وتعتبر من وسائل المقاومة الحديثة التى تعرف باسم المقاومة المستحثة وذلك عن طريق تحفيز أو حث جزء من أجزاء النبات لدفعه على مقاومة الأمراض بإستخدام وسائل متنوعه مثل الحث الفيزيائى عن طريق إستخدام الأشعه فوق البنفسيجية أوأشعة جاما.
ومن هنا جاء إستخدام هذه التقنية الجديدة في مجال زراعة الأنسجة النباتية والتي تعتمد على حث البراعم الداخلية على التوالد والتكاثر بدون تكوين الكالس وإنتاج شتلات خالية من الأمراض النباتية مع تجانس النباتات فى النمو والحصول على أعداد كبيرة من النباتات فى أقل وقت، حيث أمكن تطبيقها بنجاح عن طريق إستخدام أشعة جاما ونقلها لمحاصيل مختلفة كالطماطم (Solanum lycopersicon L.) والهيبسكس (Rosa-sinensis L.) للمرة الأولى فى مصر لمقاومة أمراض الفيتوبلازما المختلفة وتقييم جرعات مختلفة من أشعة جاما لتحديد الجرعات المناسبة لكل نبات وكذلك إختيار الأوساط الغذائية المناسبة لزراعة الأجزاء النباتية المشععة وذلك فى معمل زراعة الانسجة بقسم بحوث الفيروس والفيتوبلازما بمعهد بحوث أمراض النباتات – مركز البحوث الزراعية بالجيزة.
أن تطبيق أدوات جديدة تعتمد على تقنية زراعة الأنسجة والتشعيع باستخدام أشعة جاما قد تساعد على إقتراح استراتيجيات فعالة لمقاومة الأمراض النباتية بصفة عامة وأمراض الفيتوبلازما بصفة خاصة وكذلك منع إنتشارها بالإضافة إلى تحقيق التنمية الزراعية ودفع عجلة النمو والتقدم الزراعى عالمياً وزيادة الصادرات عن طريق تحسين إنتاجية المحاصيل المختلفة وزيادة كمية الغذاء التى ينتجها النبات الواحد وتقليل الفقد فى المحصول نتيجة الإصابة بالأمراض وذلك للايفاء بالمتطلبات المتزايدة للبشرية نتيجة الزيادة الهائلة فى أعداد السكان وتقليل معدلات الإستيراد واخيراً تحقيق الاكتفاء الذاتى.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
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In silico drugs analogue design: novobiocin analogues.pptx
Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture techniques
1. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/314626971
Ultrastructural changes in tomato plant induced by phytoplasma infection and
attempts for its elimination using tissue culture techniques
Article · December 2016
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Occurrence, Etiology and Molecular Characterization of Phytoplasma Diseases on Solanum lycopersicum Crop in Egypt View project
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Plant Pathology Research Institute
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2. Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Correspondence: dr.samahmokbel@gmail.com
Ultrastructural changes in tomato plant induced by phytoplasma infection
and attempts for its elimination using tissue culture techniques
Eman A. Ahmed1
, Osama Y. shalaby2
, Emad F. Dwidar2
,
Samah A. Mokbel1
and Ahmed K. El-Attar1
1Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center (ARC), P.O.
Box 12619, Giza, Egypt.
2Plant Pathology Department, Faculty of Agriculture, Fayoum University, P.O. Box 63514, Fayoum, Egypt.
ABSTRACT:
Light microscopy was used to compare the anatomical characteristics of stem, flower petiole and leaves
of infected tomato with phytoplasma with healthy ones in order to reveal anatomical modifications caused
by the infection process. The results obtained showed that infection of tomato plants with phytoplasma
led to an increase in stem diameter by 10.23% as well as greatly increase in measurements of the other
stem components while the diameter of pith was decreased by 38.46%. This infection was led to an
increase in the diameter of petiole by 109.2% and also the other components of flower petiole. At the
same time, tomato leaves were greatly affected as a result of the infection with phytoplasma. The
thickness of leaf blade was remarkably increased by 200% as well as thickness of either palisade or
spongy tissues by 212.5% or 275%, and significant malformation in leaflet midvein was observed which
consequently led to increase in both length and width of midvein by 15.19% and 5%. Electron
microscopy was used to recognize the internal changes in cell organelles due to phytoplasma infection.
The results obtained showed that, general disorganization of phloem tissue and thickness of cell wall
resulted from high concentration of phytoplasma units; necrosis starts in companion cells; severe damage
in chloroplasts with abnormal membrane and thylakoid system was absent. The xylem vesicles were
characterized by deforming the secondary walls. Three methods were done towards the production of
phytoplasma-free tomato plantlets through tissue culture using antibiotic compound (tetracycline
hydrochloride in three concentrations 25mgL-1, 50mgL-1 and 75mgL-1), irradiation by three doses (3, 5
and 10 Gy) of gamma ray and natural compound (1ml of garlic juice). All treatments proven to be a very
useful effect against phytoplasma, except concentration 25 mgL-1 of tetracycline hydrochloride. The
findings of this study have proven treatment at lower dose of gamma rays (3 Gy) of efficient methods on
growth promotion.
Key words: Phytoplasma; Light microscopy; Electron microscopy; Tissue culture; Anti-phytoplasmal
activity.
INTRODUCTION:
Tomato is one of the most important
vegetable crops in Egypt and considered as
number one in terms of total production and
cash value. Tomatoes are grown in three
seasons' winter, summer and autumn on
about 3 percent of Egypt's total planted area
(Baka, 2014).
Phytoplasmas (class Mollicutes,
genus Candidatus Phytoplasma) are among
the smallest bacterial plant pathogens that
caused severe malformation on infected
plants, their flowers turn into leafy shoots or
their petals into green, dwarfism (general
stunting), phyllody, virescence, rapid
senescence and flower sterility that
transform plants into zombies or unable to
produce offspring, this transformation
attracts the sap-sucking insects that carry the
pathogen to new hosts (Hogenhout et al.,
2008). Such symptoms were observed in
tomato plants during phytopathological
surveys in the main agricultural areas during
2012-2013 (Ahmed et al., 2014). Set of
diagnostic procedures and transmission
electron microscopy (TEM) technique were
performed to analyze the pathogens in
collected samples prior the molecular
techniques to confirm the results, and also to
3. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
formulate early disease management
strategies.
Plant tissue culture is a major tool of
biotechnology and of particular importance
with vegetatively propagated crops in which
infected planting materials transmit the
pathogen to the new crop or has a great role
in improving productivity of crops through
rapid availability of healthy plants to avoid
the great yield losses caused by
phytoplasmas. Micropropagation of
phytoplasma-free plants was successfully
introduced using treatment of plant tissues
with antibiotics like tetracycline
hydrocloride (Wongkaew and Fletcher,
2004; Singh et al.,2006; El-Banna et al.,
2007; Gribaudo et al., 2007; Mahrous, 2012)
or with antimicrobial substance like garlic
(Durairaj et al. 2009 and Mahrous, 2012).
Recently, radiotherapy has appeared as a
new approach for producing phytoplasma-
free plants (Mokbel and El-Attar, 2014).
The objectives of this study are: (i):
To investigate and recognize the internal and
abnormalities impacts induced by
phytoplasma infection in the tomato host
according to recent studies have shown that
the association between plants and
phytoplasmas can result in anatomical
alteration in phloem tissues of infected
plants, and great differences between
healthy and diseased samples using
microscopic examination of longitudinal,
cross or ultra-thin sections of leaf blade, leaf
petiole and stem (El-Banna et al., 2007). (ii):
To determine the efficiency of different
techniques toward production of
phytoplasma-free tomato plantlets and
mitigation of phytoplasma disease.
MATERIALS AND METHODS
Plant materials:
The plant materials selected for
present study were examined using nested
PCR and have been widely investigated in
previous study (Ahmed et al., 2014). The
molecular investigation results of previous
studies on samples collected from natural
infected tomato plants, suggest that the
presence of three infections of phytoplasma
in different areas of tomato-growing fields
in Egypt where, witches’ broom, phyllody
and big bud phytoplasma were successfully
identified, molecularly characterized and
accessioned by GenBank under accession
numbers KT225548, KT230865 and
KT225545 respectively. In the present study
we focused on the following points:
Histopathological studies:
Ligh microscopy:
Plant materials of infected tomato
(stem, flower petiole and leaflets) were fixed
and preserved in F.A.A., dehydrated,
embedded in paraffin wax, then serially
sectioned at 20-μ-thick and finally, stained
according to the conventional method (Sass,
1958) with the crystal violet–erythrosin
combination, cleared in carbol xylene and
mounted in Canada balsam. Control pieces
from healthy plant were also prepared for
comparison. The investigations were carried
at Plant Pathology Department, Faculty of
Agriculture, Fayoum University.
Electron microscopy (ultrathin-sections):
Ultra-histopathological changes due
to phytoplasma infection on the cell
components of infected tomato were studied
using electron microscopy (EM) according
to Hanschke and Schauer (1996) and carried
out in Electron Microscopy Lab, Faculty of
Agriculture, Cairo University.
Phytoplasmas elimination:
Antimicrobial agents:
One tablet (250 mg) of tetracycline
hydrochloride (C22H24N2O8.HCl), produced
by SEDICO Pharmaceutical Company
(Egypt) was used within the recommended
period. Stock solutions were prepared by
dissolving tablet using 250 ml of autoclaved
4. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
techniques
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
liquid MS medium to produce 1mg/ml
solution.
Garlic extract, prepared as a juice
from crushed garlic cloves, and then
centrifuged at 5,000 rpm for 5 min. The pure
liquid juice was carefully removed from the
top of the liquid by a syringe.
In vitro treatment processes:
The phytoplasma-infected plants
(scions) were introduced to the greenhouse,
and graft inoculated (Fig.1) to twenty
healthy tomato plants (root stocks). Stem
segments (2-3 cm long) were taken from the
plants grown under greenhouse conditions
and then surface-sterilized as described by
(Mokbel and El-Attar, 2014).
Three single node cuttings of 1.0 cm
were cultivated in culture jar containing 25
ml Murashige and Skoog (1962) medium
(full strength MS) with 30gL-1
sucrose and
8gL-l
agar. Shoots were micro-propagated
by repeatedly sub-culturing in a second
medium with the same basal composition
but without agar using filter paper bridge,
thus giving rise to stocks of plant for
different treatments. In a first treatment,
explants were cultivated on medium with
three concentration of tetracycline
hydrochloride (25, 50 and 75 mgL-1
) and
were applied separately. In a second
treatment, tomato explants were transferred
to fresh medium with liquid garlic juice (1
ml/25ml MS). Antimicrobial agents were
applied through sterilized filter 0.22 µm
Millipore onto culture medium after
autoclaving. Twenty infected explants with
phytoplasma were used per each treatment.
Control experiments free of antimicrobial
agents were also set up. In third treatment,
five jars with three explants were irradiated
with each dose 3, 5 or 10 Gy of gamma
irradiation for 30 min. five non-irradiated
culture jars were served as control treatment.
The source of gamma irradiation was 60
Co
gamma cell 3500, from the Middle Eastern
Regional Radioisotope Center for the Arab
countries, Giza, Egypt.
The cultures at all growth stages
were incubated under artificial conditions
25±1ºC, 16h photoperiod, 3000 Lux for
three weeks, and then were daily observed
and survival percentages were investigated.
The survived explants of each treatment
were sub-cultured for 3 weeks on fresh MS
medium, and then transferred onto rooting
liquid medium contain naphthalene acetic
acid (0.4 mgL-1
) for 3 weeks.
The presence of phytoplasma was
assayed by nested PCR as following: For
each condition, DNA was extracted from
treated fresh shoots as well as diseased ones
(control treatments) using the standard assay
developed by Dellaporta et al. (1983). Two
pairs of universal primers, P1/P7 and
R16F2n/R16R2 were used for the first and
second PCR, amplifying fragments of 1.8
Kb and 1.2 bp, respectively, was performed
according to the protocol described by Wang
and Hiruki (2001).
Treatment Efficiency (TE):
At the end of each in vitro treatment,
treatment efficiency (%TE) was determined
according to the rate of success in
eliminating phytoplasma and percentage of
surviving plantlets as follows:
Number of survival phytoplasma-free plants
TE = --------------------------------------------------------------------------------------------------- × 100
Total number of plants in each treatment
5. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Fig.1. Transmission of phytoplasma from donor infected tomato to receptor healthy tomato
through wedge grafting. Healthy tomato plants as root stock and the infected tomato plants as
scions (A). Symptoms of big bud and witches’ broom were developed on graft inoculated tomato
plants after 52 and 45 days from grafting, respectively (B and C). Healthy tomato plants as
control (D).
RESULTS
Histopathological studies:
Histopathological studies using light and
electron microscopy revealed that dramatic
changes occurred in anatomical structure of
tomato plants stem, flower petiole and
leaflet.
Data presented in Table (1) show that
the infection of tomato plants with
phytoplasma led to an increase of the
diameter of the stem, thickness of the cortex,
thickness of the vascular tissues and
thickness of the inner phloem tissues by
10.23%, 32.35%, 37.5% and 525%,
respectively while, the diameter of the pith
decreased by 38.46% as compared to those
in healthy plants (Fig.2[1]).
Data presented in Table (2) as well as
the cross section of the infected tomato
flower petiole (Fig.2[2]) reveal that the
infection with phytoplasma led to an
increase of the components of flower
petiole. The diameter of the petiole was
markedly increased by 109.2 %, the cortex
thickness by 120%, the thickness of the
vascular tissues by 51.89% and greatly
increases in the thickness of the inner
phloem by 333.33%.
The changes on the anatomical structure
of the infected tomato leaflet with
phytoplasma were clearly observed in
Figure (2[3]) and Table (3). The infection of
tomato plants with phytoplasma led to
increase the thickness of the leaf blade by
200% and thickness of both palisade and
spongy tissues by 212.5% and 275%,
respectively. In addition, greatly
malformation in leaflet midvein was
observed which consequently led to increase
both length and width of midvein by 15.19%
and 5% respectively. Also, the number of
the xylem vessels was remarkably increased
by 126% and both length and width of the
vascular bundle by 25% and 5.7%,
respectively.
On the other side, the investigation of
ultra-thin sections by transmission electron
microscopy were carried out to recognize
the ultrastructure of both healthy and
infected tomato plants, and resulted in
highly cytopathological changes of the
infected plants as compared with that in the
healthy cells including, general
6. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
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Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
disorganization of phloem tissue and cellular
abnormalities of the infected tomato plants
(Fig.3), thickness of cell wall and irregular
in shape, high concentration of phytoplasma
units in the sieve element of phloem cell
(Fig.4), in addition, gradual degradation and
dissocition of the adjacent cell wall followed
by complete lysis (Fig.5), necrosis starts in
companion cells (Fig.6) and changes on the
plasma membrane of phloem cells was
clearly observed as well as shattering of its
structure (Fig.7). It was also observed that
the chloroplasts of the infected cells became
malformed and large in size if compared
with healthy ones, with abnormal membrane
and thylakoid system was absent (Fig.8)
similarly, the xylem vesicles were
characterized by deforming of secondary
cell walls (Fig.9).
Table 1: Effect of phytoplasma infection on the anatomical structure of tomato stem.
Plant paramiters
Healthy
tomato
Infected
tomato
% Change
Stem diameter (µm) 5080 5600 + 10.23
Cortex thickness (µm) 340 450 + 32.35
Number of cortex layers 11 9 - 18.18
Vascular cylinder thickness (µm) 3900 4250 + 8.974
Vascular tissues thickness (µm) 800 1100 + 37.50
Xylem zone thickness (µm) 725 525 - 27.58
Number of xylem vessels 220 200 + 9.090
Pith diameter (µm) 3250 2000 - 38.46
Inner phloem zone thickness(µm) 80 500 + 525.0
Table 2: Effect of phytoplasma infection on the anatomical structure of tomato flower
petiole.
Plant paramiters
Healthy
tomato
Infected
tomato
% Change
Flower petiole diameter (µm) 1525 3190 + 109.2
Cortex thickness (µm) 125 275 + 120.0
Number of cortex layers 8 8 0
Vascular cylinder thickness (µm) 1175 2590 + 120.4
Vascular tissues thickness (µm) 370 562 + 51.89
Xylem zone thickness (µm) 220 160 - 27.27
Number of xylem vessels 100 308 - 208.0
Pith diameter (µm) 795 1500 + 88.67
Inner phloem zone thickness (µm) 90 390 + 333.3
7. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Table 3: Effect of phytoplasma infection on the anatomical structure of tomato leaflet.
Plant paramiters
Healthy
tomato
Infected
tomato
% Change
Midvein length (µm) 1020 1175 + 15.19
Midvein width (µm) 1000 1050 + 5.000
Blade thickness (µm) 100 300 + 200.0
Palisade layer thickness (µm) 40 125 + 212.5
Spongy layer thickness (µm) 40 150 + 275.0
Number of xylem vessels 23 52 + 126.0
Vascular bundle length (µm) 240 300 + 25.00
Vascular bundle width (µm) 520 550 + 5.769
Fig.2. Transections of tomato stem (1) flower petiole (2) and leaflet (3) as affected by
phytoplasma infection:
(A) Healthy plant. (B) Infected plant.
vb = vascular bundle. xv = xylem vessels. cx = cortex.
oph = outer phloem. iph = inner phloem. pi = pith.
pa = palisade tissue. sp = spongy tissue. X =70
8. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
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Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Fig.3. Transmission electron micrographs of the phloem tissues from the healthy and infected
tomato plants. Phloem tissues of the healthy tomato plant (A). Disorganization of the phloem
tissue and cell abnormalities of symptomatic tomato plants (B). X=4000.
Fig.4. Transmission electron micrographs of the phloem tissues from the healthy and infected
tomato plants. Phloem tissues of the healthy tomato plant (A). Sieve element of the phloem filled
with high concentration of phytoplasma and uneven thinking of cell wall of symptomatic tomato
plants (B). X=8000. cw = Cell wall, se = Sieve element, p = Phytoplasma units.
Fig.5. Transmission electron micrographs of the phloem tissues from the healthy and infected
tomato plants. Cell wall of the phloem cell of the healthy tomato plant (A). Gradual degradation
and dissociation of the cell wall of the infected cell (B). X=15000.
9. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Fig.6. Transmission electron micrograph of the phloem tissues from the healthy and infected
tomato plants. Phloem parenchyma cell of the healthy tomato plant (A). Phloem cell of
symptomatic tomato plant became necrotic (B). X=15000.
Fig.7. Transmission electron micrographs of the phloem tissues from the infected tomato plants
with phytoplasma. Plasma membrane of the phloem cell affected as its structure was distributed
(A and B). X=20000. cm = Cell membrane, v = Vacuole, p = Phytoplasma units.
Fig.8. Transmission electron micrograph of the phloem tissues from the healthy and infected
tomato plants. Chloroplast (ch) of the phloem parenchyma cell of healthy tomato plant (A).
Phloem parenchyma cells of symptomatic tomato plant showing malformed chloroplast (B).
X=30000.
Fig.9. Transmission electron micrograph of the xylem vesicles from the healthy and infected
tomato plants. The xylem vesicles of the healthy tomato plant (A). Deformation of secondary
wall development on xylem vesicles of symptomatic tomato plant (B). X=4000
10. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
techniques
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Controlling of phytoplasma disease:
Data presented in Table (4) indicated
that the percentage of phytoplasma-free
tomato plantlets was achieved by increasing
the concentration of tetracycline
hydrochloride in culture medium. These
percentages were 22.2% and 70.5% upon
using 50 and 75 mgL-1
tetracycline
hydrochloride with the highest percentages
obtained 90% and 85% of survival
respectively, as compared with control 0%.
While, the addition of the tetracycline
hydrochloride at a concentration of 25 mgL-
1
, had no apparent effect on eliminating of
phytoplasma. A concentration of 75 mgL-1
,
was found optimal in this study (Fig.10),
based on the detection for the presence or
absence of phytoplasma by PCR (Fig.11).
On the other side, three different doses
of gamma radiation 3, 5 and 10 Gy (for 30
min in each) were investigated on
controlling phytoplasma, survival rate and
development of tomato shoots. Data
presented in Table (5) demonstrated that the
percentage of phytoplasma-free shoots as
determined by nested PCR and the number
of survival shoots after been submitted to
radiation treatment varied with the dose of
treatment applied where, the best culture
evolution (Fig.10) and survival rate (66.6%)
obtained with 3 Gy dose, but their treatment
with 5 Gy dose reduced the survival rate to
53.5% with growth retardation. Reduction
was particularly evident in stem cuttings that
were subjected to 10 Gy which led to the
loss of a large number of explants being
73.4% and only four plantlets still survived.
Regarding the various treatments, the
nested PCR using the universal
phytoplasma-specific primers conducted
after re-cultured of the treated explants on
fresh liquid medium and at the end of
incubation period. The results confirmed the
absence of phytoplasmas in all tested shoots
regenerated from stem cuttings while
showed a clear band at the specific size 1200
bp only when the template DNA was
extracted from the non-irradiated tomato
samples (Fig.12).
Finally, data in Table (6) demonstrated
that the treatment with concentrated garlic
juice (1ml/jar) was more distinguished and
optimal for eliminating phytoplasma in the
present study where increased the
percentage of phytoplasma-free plantlets to
87.5% where a total number of 16 tomato
plantlets were tested for the presence or
absence of phytoplasma by PCR and 14
tomato plantlets did not yield any fragment
specific for phytoplasma infection (Fig.11).
In addition, the fourteen tomato plantlets
propagated well in subsequent sub-cultures
(devoid of garlic) without any phytoplasma
symptomatic (Fig.10).
Treatment Efficiency (%TE):
According to the treatments and data
demonstrated in tables (4,5,6), the in vitro
treatment with natural product proved to be
more effective (70%) in our study in terms
of controlling phytoplasma and the high
percentage of survival rate followed by
irradiation with dose of 3 Gy of gamma ray
(66.6%) and finally a concentration 75 mgL-
1
of tetracycline hydrocloride (60%).
However, our results showed that the
treatment with dose of 3 Gy of gamma
irradiation had a positive effect on viability
and growth ability of tomato plantlets
(Fig.10).
Rooting stage:
All phytoplasma-free plantlets from
those efficient treatments were sub-cultured
on liquid rooting medium contain 0.4 mgL-1
of NAA. The results found that roots
initiated within few days. With time, well
developed and long intensive roots were also
formed at the end of incubation period
(Fig.13).
11. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Table 4: Influence of different concentrations of tetracycline hydrochloride on the
percentage of survival rate and phytoplasma-free tomato plantlets.
Tetracycline
hydrochloride
concentration
S %S
PCR
detection
Phytoplasma free
plantlets %
%TE
In H
25 mgL-1
16 80 16 0 0 0
50 mgL-1
18 90 14 4 22.2 20
75 mgL-1
17 85 5 12 70.5 60
0 (Control) 15 75 15 0 0 -
Twenty Plantlets per each treatment, In= Infected plantlets, H=Healthy plantlets,
S=Survival explants. TE= Treatment Efficiency.
Table 5: Effect of different doses of gamma radiation on the percentage of survival rate
and phytoplasma-free tomato plantlets.
Radiation
dose (Gy)
S %S
Phytoplasma free
plantlets %
%TE
3 10 66.6 100 66.6
5 8 53.5 100 53.5
10 4 26.6 100 26.6
0 (Control) 12 80 0 -
Data are based on PCR assay, 15 Plantlets per each treatment, S=Survival explants.
TE= Treatment Efficiency.
Table 6: Influence of liquid garlic juice on the percentage of survival rate and
phytoplasma-free tomato plantlets.
Garlic
treatment
S %S
PCR
detection
Phytoplasma free
plantlets %
%TE
In H
1ml/
25ml MS
16 80 2 14 87.5 70
0 (Control) 17 85 17 0 0 -
Twenty Plantlets per each treatment, In= Infected plantlets, H=Healthy plantlets,
S=Survival explants. TE= Treatment Efficiency.
12. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
techniques
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Fig.10: In vitro treatment. (A): Treatment of the infected shoot tips of tomato plants with
antibiotics compound (tetracycline hydrochloride), radiotherapy (gamma ray) and antimicrobial
substance (garlic juice) from left side, respectively. (B): Healthy in vitro plantlets developed
after treating with 75 mgL-1
of tetracycline hydrochloride, dose of 3 Gy of gamma irradiation
and 1ml of garlic juice from left side, respectively.
Fig.11: Electrophoresis analysis for the PCR products amplified from treated in vitro tomato
plants with tetracycline hydrochloride and garlic juice. C: None treated infected tomato plant. H:
Healthy tomato plant. GT: infected tomato plants treated with 1ml of garlic juice. L1, L2 and L3:
infected tomato plants treated with 25, 50 and 75 mgL-1
of tetracycline hydrochloride,
respectively. M: 1 Kb DNA Ladder.
13. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
Fig.12: Electrophoresis analysis for the PCR products amplified from irradiated and none
irradiated tomato plants. C: None irradiated infected tomato plant. L1, L2 and L3: irradiation
treatments of in vitro explants with 3, 5 and 10 Gy doses of gamma ray. HPC: Healthy plant as
control. M: 1 Kb DNA Ladder.
Fig.13: Roots development from healthy tomato plantlets at the end of incubation period (21
days) from efficient treatments with 75 mgL-1
of tetracycline hydrochloride (A), dose of 3 Gy of
gamma ray (B) and 1ml of garlic juice (C). Roots development from infected tomato explants, as
control (D).
DISCUSSION
In this research work, the aim was to
investigate and recognize the potential
damage induced by phytoplasma infection by
studying histopathological and cytological
changes in tomato host, and also to elucidate
the possible mechanism of phytoplasma
elimination through tissue culture techniques.
Light and electron microscopic
examination on the effect of phytoplasma
infection on anatomical and ultrastructural
changes in tomato plants revealed
deterioration effects on stem, flower petiole
or leaflets tissues, and important histological
variations included general disorganization
and deformation of phloem tissue, that
mainly due to the adhesion of phytoplasma
units with the inner surface of the cell plasma
14. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
techniques
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
membrane that represent a functional aspect
of the phytoplasma which uses the sterols of
the cell membrane in order to satisfy their
energy needs for growth and division
(Christensen et al., 2005 and Mou et al.,
2013).
The obtained results through the
investigations into the fine structure of sieve
tube contents in the present study agree with
that reported by El-Banna and El-Deeb
(2007) and Randall et al. (2011) who found
that considerable differences between healthy
and malformed samples (leaf blade, leaf
petiole and stem) and the most important
changes are disorganization of phloem cells
that accompanied with an increase in cell
wall thickness, middle lamella and size of
spaces between cells, that may be due to the
accumulation of sugars and starch
concentration as a result of phloem
malformation which, remarkably affected in
translocation of carbohydrates and other
photosynthesis molecules.
Similarly, the histopathological and
cytological changes of infected tomato plants
with phytoplasma like thickening of cell wall
or irregular shape, malformation of
chloroplasts, deformation of the xylem
vesicles and the changes on the plasma
membrane of phloem cells as well as
shattering of its structure, have been reported
by other authors (Carpita and Gibeaut 1993,
Siddique et al. 1998, El-Banna et al. 2007
and MacLean et al. 2011).
Further, Esau (1977) reported that
infection with phytoplasma led to anatomical
alteration in spinach phloem tissues such as
phloem degeneration and necrosis of sieve
tubes as well as abnormal cell proliferation.
Three methods were successfully done
towards the production of phytoplasma-free
tomato plantlets through tissue culture using
antibiotic compound (tetracycline
hydrochloride), gamma ray and natural
product (garlic juice), the results indicated
that in vitro treatment with tetracycline that
routinely used for controlling of different
strains of phytoplasma led to a strong
recovery of infected tomato explants
especially with concentration 75 mgL-1
used
in the present study as evidenced by the high
percentage of elimination (70.5%) and the
survival percentage of explants (85%), which
may be due to inhibits protein synthesis in
microorganisms (El-Banna et al., 2007) and
thus inhibit the growth or replication of
phytopalsma. Also, Mahrous (2012) has
shown that the tetracycline proved to be
effective in eliminating phytoplasma from
70% infected tomato plantlets using 75mgL-1
,
and also failed to eliminate phytoplasma
using 25mgL-1
. Similarly, Singh et al. (2006)
was reported that 50% of infected plants
cultivated on MS media and subjected to
concentration of 75 mgL-1
of an
oxytetracycline for two weeks were
phytoplasma free, and they remained healthy
for more than 3 years. In general, several
investigators reported that adding of
tetracycline to the culture media allowed
permanent phytoplasma elimination as well
as other microorganism, highly sensitized to
tetracycline antibiotics, like Spiroplasma
(Saglio et al., 1973 and Davies and Clark
1994).
Obtained results through tissue culture
technique coupled with radiotherapy proved
that gamma ray had a positive effect on
phytoplasma elimination. In vitro treatment
with different doses of gamma irradiation (3,
5 and 10 Gy) have been successfully
controlled phytoplasma especially, dose 3 Gy
as evidenced by the high percentage of
elimination (100%) and the survival
percentage of explants (85%).
The successful controlling of
phytoplasma using gamma ray are supported
also by Mokbel and El-Attar (2014) who
completely succeeded in eliminating witches’
broom phytoplasma with percentage 100%
using various doses (5, 10, 15, 20 and 25 Gy)
15. Eman et al.
Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
of gamma ray with survival percentages (96,
73.3, 73.3 20 and 13.3%, respectively) from
infected hibiscus plantlets through tissue
culture technique. Moreover, several
investigators reported that gamma irradiation
led to successful inactivation of some plant
viruses like citrus tristeza virus (CTV),
necrotic ring spot virus (NRSV) and prune
dwarf virus (PDV) (Megahed and Moore,
1969 and Ieki and Yamaguchi, 1984). Also,
consistent with many studies in previous
years, have shown that irradiation as effective
means of controlling human pathogens such
as Escherichia coli (Thayer and Boyd, 1993)
as well as food-borne pathogens (Beuchat,
1996 and Sumner and Peters, 1997) and the
fungal pathogens (Rhizoctonia solani Kuhn
and Sclerotium rolfsii Sacc) that attacked
sugar beet plant (Moussa and Rizk 2003).
Moreover, gamma irradiation has been also
used to sterilize agricultural products in order
to increase their conservation time or to
reduce pathogen when being traded from a
country to another (Melki and Salami, 2008).
The successful treatment with gamma
ray in the current study may be a quite similar
to mechanism of gamma ray during
sterilization processes for the control of food
contaminating microorganisms like bacteria,
however it would not be a sterilizing step as it
is for bacteria that has a cell wall and are ten
times larger than phytoplasma, and the
presence of cell wall is a step for irradiation
resistance, taking in account the
phytoplasmas are very small prokaryotes
which are related to bacteria, but in contrast
to bacteria, they do not have a cell wall and
thus collapsed with the effect of high-energy
gamma photons (1.33 MeV) given off by
Cobalt-60. This energy can penetrate plant
cell and hit and break down the double helix
of the pathogen-DNA or split the water
molecules and generate free hydrogen (H+
),
hydroxyl (OH-
) and oxygen (O-2
) radicals that
capable of killing the pathogen by
deactivating and damage pathogen-DNA,
which in turn causing defects in the genetic
instructions and disrupting its function and is
therefore inhibiting pathogen reproduction
through a safe and nontoxic treatment,
without any radioactivity even if the high
doses are used up to 10 kGy (Who, 1981 and
Mokbel and El-Attar, 2014).
On the other side, the influences of
irradiation on plant growth and development
depend mainly on doses of gamma irradiation
used in the present study, and completely
consistent with many studies carried out on
the stimulation and inhibition of plant growth
by applying gamma irradiation where,
increasing the doses of gamma irradiation
lead to severe effects on the plant
development and the surviving plants
percentage, which decreased linearly in our
study with increasing doses of gamma
irradiation that may be attributed to the
inhibition of DNA synthesis, destruction of
the membrane system of mitochondria and
chloroplasts, or other physiological damages
and complications after irradiation
treatments, like disruption of protein
synthesis, hormone balance, enzyme activity
(Ladanova, 1993; Kovacs and Keresztes
2002; Wi et al., 2006; El Sherif et al., 2011;
Shekari, et al., 2011; Hasbullah et al., 2012
and Minisi et al., 2013).
Concerning garlic juice effect on the
percentage of phytoplasma-free plants; the
concentration 1 mg/l increased the percentage
to 87.5%, as compared with the control.
Similarly, Mahrous (2012) mentioned that
using grinded garlic bulbs in distilled water
by a rate of (1:2 w/v) inhibited witches’
broom phytoplasma from 83.3% infected
tomato plants. Furthermore, Durairaj et al.
(2009) and Olaiya et al. (2011) reported that
the raw juice of garlic was effective against
many strains of pathogenic bacteria that have
become resistant to antibiotic, and attributed
the antibacterial activity of garlic bulbs to
allicin where interferes with RNA production
16. Ultrastructural changes in tomato plant induced by phytoplasma infection and attempts for its elimination using tissue culture
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Egyptian J. Virol., Vol. 13 (1): 34- 51, 2016.
and lipid synthesis and cause damage of cell
membranes.
In conclusion, the same procedures of
nested polymerase chain reaction (Nested-
PCR), are successfully carried out for the
detection of phytoplasma in the current study
as our previous study (Ahmed et al. 2014),
but the main difference between these two
studies: the samples used in current research
came from tissue cultured plants, while the
samples used in previously came from natural
infection and experimentally inoculated
samples. Also, our results demonstrated that
the EM technique is a highly valuable in
detection of phloem inhabiting pathogens,
and is a very important tool in correct
diagnosing of phytoplasma diseases prior the
molecular techniques to prove the results in
addition to the anatomical features represent
a reliable source of information about the
effect of phytoplasma on the nature of
different organs of tomato plant (stem, flower
petiole and leaflet). The use of new tools
based on tissue culture technique and gamma
irradiation for controlling of phytoplasma
diseases may help to propose effectively
strategies for obtaining healthy planting
material and prevent further their prevalence.
Received: Oct. 2015; Accepted: Dec. 2015;
Published: Jan. 2016
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