This document describes a study that established an efficient protocol for in vitro regeneration and genetic transformation of Pelargonium graveolens (geranium). Maximum callus proliferation was obtained on medium supplemented with combinations of 20 μM IBA + 10 μM kinetin, 20 μM IBA + 10 μM BAP, or 20 μM IAA + 10 μM kinetin. Plantlets were regenerated from callus under 16 hours of light per day. The regenerated plantlets were successfully acclimatized and transferred to soil. Genetic transformation of geranium was demonstrated using Agrobacterium tumefaciens containing a binary vector.
A pot experiment was conducted to evaluate the nematicidal efficacy of a biocontrol agent, Pseudomonas flourescens for the management of root-knot nematode, Meloidogyneincognita on chickpea (Cicer arietinum L.) cv.‘Avarodhi’ under glasshouse conditions. All the treatments were found to significantly improve the growth and physiological parameters of chickpea and reduction in pathological parameters as compare to untreated inoculated control. The highest improvement was observed in those plants treated with P. flourescens alone. Concomitant and sequential inoculation of P. flourescens with M.incognita also showed significant improvement in growth parameters of chickpea. Least enhancement in growth parameters was observed in those plants inoculated with nematode alone. It may be due to the nematcidal behaviour of P. flourescens against root-knot nematode, M. incognita. Hence, it may be concluded that P. flourescens as biocontrol agent is better substitute against chemical nematicides for the sustainable management of M. incognita and reduce environmental hazards.
Standardization of punica granatum explant and callus induction through micro...eSAT Journals
Abstract Pomegranate (Punica granatum L.) and variety name ‘Bhagwa’ is an ancient, important fruit crop in India and in subtropical countries of the world as it possess various pharmaceutical and therapeutic properties. This is subjected to bacterial blight caused by Xanthomonas axonopodis pv causing a huge loss of about 50-100% in production. In order to develop a disease resistant pomegranate variety, micro propagation is necessary. The different explants such as leaves, nodes, apical shoot and petals were selected. The explants were passed through surface sterilization process and found that the mortality rate was least with the apical shoots as explants when compared to other explants. Callus Initiation was done with several treatments and the percentage of callus growth was identified using one way ANOVA by which variance was tested using Fischer’s F test and LS (Least Squares) means by Duncan’s multiple range test which proved that the LS means was higher for all the explants those undergone MS + Sucrose (30g/l) + Adenine sulfate – 40mg/l + 6BAP – 5 mg/l treatment, specifically apical shoot explants showed 92% callus growth than other explants. The elimination of polyphenol exudation was successful with silver nitrate of 5 mg/l which eradicated the browning of the tissues and paved way for the regeneration of the shoots. Key Words: Bhagwa, Micropropagation, Apical shoots, Callus induction, ANOVA, Duncan’s test, Polyphenol exudation
Bioactivity of Locally Available Plants on Cotton Whitefly, Bemisia tabaci an...IJEAB
Aqueous, diethyl ether, chloroform, petroleum ether, N-hexane and benzene extracts of locally available plant species were tested for phytochemical and insecticidal bioactivity against cotton whitefly, Bemisia tabaci, under controlled conditions. This study is within bioprospection context, for utilizing local plant species as alternative in sustainable agriculture development. The leaf and stem extract was used. The whole plant extract of T.procumbens followed by N.oleander and V.rosea showed repellent and toxic effect against adult and second nymphal instars. Leaf extract of all three plants showed high inhibition activity against nymphal instars. In case of flower extract less inhibition activity was shown respectively. Fungi which grow on the cotton plant was screened, characterized and checked for antifungal activity against the extracts of the plant material. Phytochemical analysis was also carried out by standard protocols.
Propagation of pomegranate (Punica granatum L.) by tissue culture Abdul Hakim Salehi
Seminar Presented by Abdul Hakim Salehi,
Sr. MSc.(Hort) Fruit Science Department
College of Horticulture Bengaluru,
University of Horticultural Sciences Bagalkot
A pot experiment was conducted to evaluate the nematicidal efficacy of a biocontrol agent, Pseudomonas flourescens for the management of root-knot nematode, Meloidogyneincognita on chickpea (Cicer arietinum L.) cv.‘Avarodhi’ under glasshouse conditions. All the treatments were found to significantly improve the growth and physiological parameters of chickpea and reduction in pathological parameters as compare to untreated inoculated control. The highest improvement was observed in those plants treated with P. flourescens alone. Concomitant and sequential inoculation of P. flourescens with M.incognita also showed significant improvement in growth parameters of chickpea. Least enhancement in growth parameters was observed in those plants inoculated with nematode alone. It may be due to the nematcidal behaviour of P. flourescens against root-knot nematode, M. incognita. Hence, it may be concluded that P. flourescens as biocontrol agent is better substitute against chemical nematicides for the sustainable management of M. incognita and reduce environmental hazards.
Standardization of punica granatum explant and callus induction through micro...eSAT Journals
Abstract Pomegranate (Punica granatum L.) and variety name ‘Bhagwa’ is an ancient, important fruit crop in India and in subtropical countries of the world as it possess various pharmaceutical and therapeutic properties. This is subjected to bacterial blight caused by Xanthomonas axonopodis pv causing a huge loss of about 50-100% in production. In order to develop a disease resistant pomegranate variety, micro propagation is necessary. The different explants such as leaves, nodes, apical shoot and petals were selected. The explants were passed through surface sterilization process and found that the mortality rate was least with the apical shoots as explants when compared to other explants. Callus Initiation was done with several treatments and the percentage of callus growth was identified using one way ANOVA by which variance was tested using Fischer’s F test and LS (Least Squares) means by Duncan’s multiple range test which proved that the LS means was higher for all the explants those undergone MS + Sucrose (30g/l) + Adenine sulfate – 40mg/l + 6BAP – 5 mg/l treatment, specifically apical shoot explants showed 92% callus growth than other explants. The elimination of polyphenol exudation was successful with silver nitrate of 5 mg/l which eradicated the browning of the tissues and paved way for the regeneration of the shoots. Key Words: Bhagwa, Micropropagation, Apical shoots, Callus induction, ANOVA, Duncan’s test, Polyphenol exudation
Bioactivity of Locally Available Plants on Cotton Whitefly, Bemisia tabaci an...IJEAB
Aqueous, diethyl ether, chloroform, petroleum ether, N-hexane and benzene extracts of locally available plant species were tested for phytochemical and insecticidal bioactivity against cotton whitefly, Bemisia tabaci, under controlled conditions. This study is within bioprospection context, for utilizing local plant species as alternative in sustainable agriculture development. The leaf and stem extract was used. The whole plant extract of T.procumbens followed by N.oleander and V.rosea showed repellent and toxic effect against adult and second nymphal instars. Leaf extract of all three plants showed high inhibition activity against nymphal instars. In case of flower extract less inhibition activity was shown respectively. Fungi which grow on the cotton plant was screened, characterized and checked for antifungal activity against the extracts of the plant material. Phytochemical analysis was also carried out by standard protocols.
Propagation of pomegranate (Punica granatum L.) by tissue culture Abdul Hakim Salehi
Seminar Presented by Abdul Hakim Salehi,
Sr. MSc.(Hort) Fruit Science Department
College of Horticulture Bengaluru,
University of Horticultural Sciences Bagalkot
Anti-Oxidant and Antimicrobial Studies of Tinospora cordifolia (Guduchi/Giloy...SUS GROUP OF INSTITUTIONS
Plants produce a diverse range of bioactive molecules, making them a rich source of
different types of medicines and healing properties. The present study was aimed to
evaluate the anti-oxidant and antimicrobial properties of stem and root of T. cordifolia.
Total phenolic contents of different solvent extracts were determined and found that ethanol
extract had the highest phenolic content of 0.3213 mg g-1. Antioxidant assays were also
carried out by using different in vitro models such as total reducing power, hydrogen
peroxide scavenging activity assay and hydroxyl redical scavenging activity. The Ethanol
extract showed the highest total antioxidant activity. The H2O2 scavenging and hydroxyl
free radical scavenging activity was maximum 87.2 % and 91.0% found in case of ethanolic
steam extract respectively. The antimicrobial activity of ethanolic and methanolic extract of
root and stem of T. cordifolia were also evaluated against some pathogenic microorganisms
viz. E. coli, B. subtilis, A. niger and Candida sp. it was found that the various concentration
of extract viz. 50, 100, 150 and 200 mg ml-1 were tested. It was observed that the
increasing in concentration there was also increasing in antimicrobial activity reveled by
increase in size of zone of inhibition. The methanolic stem extract exhibits highest
antimicrobial activity against all four pathogens. The study shown that the extract of T.
cordifolia has a wide range of anti-oxidant as well as antimicrobial activity against bacterial
as well as fungal pathogens.
ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL SCREENING OF NEEM LEAVES AND LEMON G...IAEME Publication
In this study, the antimicrobial activity and phytochemical constituents of neem
leaves and lemon grass oil extracts were evaluated. Oil extracts of neem leaves and
lemon grass were obtained by solvent extraction method using hexane and ethanol.
Antimicrobial activity screening of plants’ oil extracts were conducted using agar well
diffusion method and the oil extracts were tested against three gram negative bacteria
(Pseudomonas aeruginosa, Klebsiella specie, Escherichia coli), one gram positive
bacteria (Staphylococcus aureus) and two fungi (Candida albicans, Rhizopus specie).
Phytochemical components of the ethanolic oil extracts were anthocyanin and
betacyanin; quinones; terpenoids and acid for lemon grass. In addition to other
phytochemicals present in lemon grass ethanolic oil extract, neem ethanolic oil extracts
tested positive to flavonoids. Lemon grass oil extract shows high activity against
Escherichia coli, Staphylococcus aureus and Candida albicans which are
representative of the three categories of microorganisms considered. Neem leaves oil
extracts have relatively low activity against most of the selected microorganisms.
Phytochemical screening and antimicrobial activity evaluation of aqueous and ...Premier Publishers
Azadirachta indica Juss (neem) is a plant which has been used for a long time as traditional medicine for household remedy against various human ailments from antiquity. To evaluate the scientific basis for the use of Azadirachta indica, both aqueous and ethanolic extracts of the dried leaves of the plant were subjected to phytochemical screening and determination of anti-microbial activity on six different species of bacteria and a fungus. The phytochemical screening of the aqueous and ethanolic extracts of dried powdered leaves of the plant was done using standard methods. The antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of zone of inhibition against the microorganisms using agar well diffusion method. The Phytochemical screening of the test plant revealed the presence of saponins, alkaloids, cardiac glucosides, phenols, resins, tannins, terpenes and steroids. Although, both plant extracts had antimicrobial effects against the test organisms, the aqueous extracts were found to show greater anti-microbial effect than ethanolic extract. Thus, the mean diameter zones of inhibition ranged from 0.03mm-40.00mm for aqueous extract and 0.50mm-21.00mm for ethanolic extract at the highest concentration of 50mg/ml. The finding of this study supports the use of neem leaf in the treatment of various microbial infections by alternative systems of medicine.
Effect of environmental pollution on the quality of an edible plant Alternant...Premier Publishers
The present study is the comparative analysis of phytochemical constituents and microbial load of an edible plant Alternanthera philoxeroides (Mart.) Griseb collected from unpolluted and polluted site. Preliminary phytochemical analysis was performed with acetone, aqueous, chloroform, ethanol and petroleum ether extracts (unpolluted and polluted site) of A philoxeroides that showed the presence of alkaloids, carbohydrates, saponins, phenols, flavonoids, aminoacids, diterpenes, tannin, terpenoids, protein, steroid, oxalate, coumarin and quinones. The ethanol extract showed higher number of phytochemical constituents when compared to the other extract of unpolluted site. The microbial load is also enumerated in the unpolluted and polluted site. In conclusion, phytochemical analysis revealed the presence of many phytoconstituents in ethanol extract and the microbial load is less in the unpolluted site when compared to the polluted site.
Invitro Study of Antibacterial Activity of Leaf and Root Extract of Rauvolfia...paperpublications3
Abstract: In this study Methanolic and chloroform leaf and root extract of Rauvolfia serpentina was studied for its antibacterial activity. Antibacterial activity of leaf and root extracts was assessed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumonia by disc diffusion method. Methanolic extract of root was showed the maximum zone of inhibition for all test organisms than the leaf extract. According to observations of root extract of 50µl/ml concentration 15.4mm, 16.2mm, 12.3mm,10.1mm and 15.0mm zones of inhibition and for concentration of 100µl/ml 22.5mm, 23.1mm, 15.1mm, 18.0mm, 22.0mm zones of inhibition were formed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumonia respectively. 50µl/ml concentration of leaf and root chloroform extracts showed no zone of inhibition against Staphylococcus aureus and Bacillus subtilis, maximum zone of inhibition was observed 15.0mm and 15.5mm against E. coli for leaf and root chloroform extract respectively. 100µl/ml concentration showed maximum zone of inhibition against all test organisms for both leaf and root extracts. All the bacteria were more susceptible to methanolic extracts than the chloroform extracts.
In-Vitro Evaluation of selected Fungicides on the Growth and Sporulation of A...Agriculture Journal IJOEAR
Broad bean (Vicia faba L.) is an important leguminous cold season crop cultivated widely in different parts of the world and in India. This crop is grown especially in U.P., Bihar, Punjab, Haryana and in the foot hill ranges of Himalayan region including north eastern states. In Manipur, it is an important winter vegetable cum pulse crop. However, this crop suffers attack of various diseases of fungi, viruses and nematodes resulting in substantial reduction in yield. Hence, an in-vitro evaluation of selected fungicides on the Growth and Sporulation of Alternaria alternata causing blight disease of broad bean (Vicia faba L.) was under taken in the present investigation. A judicious application of Tricyclazole and Copper oxychloride at 1000ppm can effectively manages the blight disease of broad bean and prevent economic loss due to disease condition.
USE OF NEEM PRODUCTS FOR THE MANAGEMENT OF INSECT PESTS OF VEGETABLESSamar Biswas
The main focus of the study to highlight the benefit of managing insect pests with neem products, based on the finding of several secondary sources. Insect management by neem is a house hold word but still it is not a common place event. Neem tree, Azadirachta indica; is a medicinal plant. The trees contain many chemical compounds of which neem seed contain much azadirachtin. It possess medium to broad spectrum of action against insects selectively with low mammalian toxicity. Azadirachtin seems to be an "ecdysone blocker. The interferences of the azadirachtin is too insect’s specific against chemoreceptor and endocrine control systems, by which it affect insect growth, feeding and oviposition. Those are so different from the mammalian system that no toxic effects have been found even after application of gram amounts of azadirachtin per kilogram of body weight. When the neem seed oil is correctly formulated its efficiency increases many times, without any adverse effect on our ecology and environment.
A high frequency microcloning protocol for subsequent cryopreservation in Kae...iosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Production of Haploids Plants from Anther Culture of Musa Paradisiaca cv. ‘Pu...RSIS International
Haploid plants were regenerated from the anther callus of banana Musa paradisiaca (AB) cv. Puttabale. The highest frequency of callus induction (90%) was observed at the concentration of 3mg/l 2, 4-D . After 20 days of incubation organization of embyroids were organised from the callus mass. Interaction of 4mg/l BAP and 0.4 mg/l IAA provoked shoot growth of the embryoids and well organised roots were developed at the concentration of 0.6 mg/l NAA and the media was agumented with 0.2% activated charcoal. Flow cytometry study was carried out to analyse the DNA content of the regenerated haploid plants. The results of the investigation reported the efficient production of haploid plants from the anther culture.
ABSTRACT- Medicinal Plants have been practiced for hundreds of centuries by tribes all over the world. From the earliest times until the end of nineteenth century plants are still the common source of medicinal treatment yet. Using natural, plant-derived medicines that are “healthier” then prescription drugs derived from synthesized products is something that appeals to consumers. The medicinal plants are of great importance because there are utilized as medicines. Aim of this research work was to evaluate the antibacterial activity of Skimmia laureola plant against various patho-genic strains of bacteria. The hot and cold water extract of Skimmia laureola were used against four bacterial strains Escherichia coli,Bacillus subti-lus, Staphylococcusaureus and Proteus mirabilis in order to check the antibacterial activity of Skimmia laureola. Antibacterial activity was conducted by agar well diffusion method. The Skimmia laureola showed different level of antibacterial activity. The hot and cold water extract of Skimmia lau-reola showed antibacterial activity against the micro-organism but not too maximum. Keywords: Medicinal Plants, Skimmia Laureola, Antibacterial Activity.
Anti-Oxidant and Antimicrobial Studies of Tinospora cordifolia (Guduchi/Giloy...SUS GROUP OF INSTITUTIONS
Plants produce a diverse range of bioactive molecules, making them a rich source of
different types of medicines and healing properties. The present study was aimed to
evaluate the anti-oxidant and antimicrobial properties of stem and root of T. cordifolia.
Total phenolic contents of different solvent extracts were determined and found that ethanol
extract had the highest phenolic content of 0.3213 mg g-1. Antioxidant assays were also
carried out by using different in vitro models such as total reducing power, hydrogen
peroxide scavenging activity assay and hydroxyl redical scavenging activity. The Ethanol
extract showed the highest total antioxidant activity. The H2O2 scavenging and hydroxyl
free radical scavenging activity was maximum 87.2 % and 91.0% found in case of ethanolic
steam extract respectively. The antimicrobial activity of ethanolic and methanolic extract of
root and stem of T. cordifolia were also evaluated against some pathogenic microorganisms
viz. E. coli, B. subtilis, A. niger and Candida sp. it was found that the various concentration
of extract viz. 50, 100, 150 and 200 mg ml-1 were tested. It was observed that the
increasing in concentration there was also increasing in antimicrobial activity reveled by
increase in size of zone of inhibition. The methanolic stem extract exhibits highest
antimicrobial activity against all four pathogens. The study shown that the extract of T.
cordifolia has a wide range of anti-oxidant as well as antimicrobial activity against bacterial
as well as fungal pathogens.
ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL SCREENING OF NEEM LEAVES AND LEMON G...IAEME Publication
In this study, the antimicrobial activity and phytochemical constituents of neem
leaves and lemon grass oil extracts were evaluated. Oil extracts of neem leaves and
lemon grass were obtained by solvent extraction method using hexane and ethanol.
Antimicrobial activity screening of plants’ oil extracts were conducted using agar well
diffusion method and the oil extracts were tested against three gram negative bacteria
(Pseudomonas aeruginosa, Klebsiella specie, Escherichia coli), one gram positive
bacteria (Staphylococcus aureus) and two fungi (Candida albicans, Rhizopus specie).
Phytochemical components of the ethanolic oil extracts were anthocyanin and
betacyanin; quinones; terpenoids and acid for lemon grass. In addition to other
phytochemicals present in lemon grass ethanolic oil extract, neem ethanolic oil extracts
tested positive to flavonoids. Lemon grass oil extract shows high activity against
Escherichia coli, Staphylococcus aureus and Candida albicans which are
representative of the three categories of microorganisms considered. Neem leaves oil
extracts have relatively low activity against most of the selected microorganisms.
Phytochemical screening and antimicrobial activity evaluation of aqueous and ...Premier Publishers
Azadirachta indica Juss (neem) is a plant which has been used for a long time as traditional medicine for household remedy against various human ailments from antiquity. To evaluate the scientific basis for the use of Azadirachta indica, both aqueous and ethanolic extracts of the dried leaves of the plant were subjected to phytochemical screening and determination of anti-microbial activity on six different species of bacteria and a fungus. The phytochemical screening of the aqueous and ethanolic extracts of dried powdered leaves of the plant was done using standard methods. The antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of zone of inhibition against the microorganisms using agar well diffusion method. The Phytochemical screening of the test plant revealed the presence of saponins, alkaloids, cardiac glucosides, phenols, resins, tannins, terpenes and steroids. Although, both plant extracts had antimicrobial effects against the test organisms, the aqueous extracts were found to show greater anti-microbial effect than ethanolic extract. Thus, the mean diameter zones of inhibition ranged from 0.03mm-40.00mm for aqueous extract and 0.50mm-21.00mm for ethanolic extract at the highest concentration of 50mg/ml. The finding of this study supports the use of neem leaf in the treatment of various microbial infections by alternative systems of medicine.
Effect of environmental pollution on the quality of an edible plant Alternant...Premier Publishers
The present study is the comparative analysis of phytochemical constituents and microbial load of an edible plant Alternanthera philoxeroides (Mart.) Griseb collected from unpolluted and polluted site. Preliminary phytochemical analysis was performed with acetone, aqueous, chloroform, ethanol and petroleum ether extracts (unpolluted and polluted site) of A philoxeroides that showed the presence of alkaloids, carbohydrates, saponins, phenols, flavonoids, aminoacids, diterpenes, tannin, terpenoids, protein, steroid, oxalate, coumarin and quinones. The ethanol extract showed higher number of phytochemical constituents when compared to the other extract of unpolluted site. The microbial load is also enumerated in the unpolluted and polluted site. In conclusion, phytochemical analysis revealed the presence of many phytoconstituents in ethanol extract and the microbial load is less in the unpolluted site when compared to the polluted site.
Invitro Study of Antibacterial Activity of Leaf and Root Extract of Rauvolfia...paperpublications3
Abstract: In this study Methanolic and chloroform leaf and root extract of Rauvolfia serpentina was studied for its antibacterial activity. Antibacterial activity of leaf and root extracts was assessed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumonia by disc diffusion method. Methanolic extract of root was showed the maximum zone of inhibition for all test organisms than the leaf extract. According to observations of root extract of 50µl/ml concentration 15.4mm, 16.2mm, 12.3mm,10.1mm and 15.0mm zones of inhibition and for concentration of 100µl/ml 22.5mm, 23.1mm, 15.1mm, 18.0mm, 22.0mm zones of inhibition were formed against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumonia respectively. 50µl/ml concentration of leaf and root chloroform extracts showed no zone of inhibition against Staphylococcus aureus and Bacillus subtilis, maximum zone of inhibition was observed 15.0mm and 15.5mm against E. coli for leaf and root chloroform extract respectively. 100µl/ml concentration showed maximum zone of inhibition against all test organisms for both leaf and root extracts. All the bacteria were more susceptible to methanolic extracts than the chloroform extracts.
In-Vitro Evaluation of selected Fungicides on the Growth and Sporulation of A...Agriculture Journal IJOEAR
Broad bean (Vicia faba L.) is an important leguminous cold season crop cultivated widely in different parts of the world and in India. This crop is grown especially in U.P., Bihar, Punjab, Haryana and in the foot hill ranges of Himalayan region including north eastern states. In Manipur, it is an important winter vegetable cum pulse crop. However, this crop suffers attack of various diseases of fungi, viruses and nematodes resulting in substantial reduction in yield. Hence, an in-vitro evaluation of selected fungicides on the Growth and Sporulation of Alternaria alternata causing blight disease of broad bean (Vicia faba L.) was under taken in the present investigation. A judicious application of Tricyclazole and Copper oxychloride at 1000ppm can effectively manages the blight disease of broad bean and prevent economic loss due to disease condition.
USE OF NEEM PRODUCTS FOR THE MANAGEMENT OF INSECT PESTS OF VEGETABLESSamar Biswas
The main focus of the study to highlight the benefit of managing insect pests with neem products, based on the finding of several secondary sources. Insect management by neem is a house hold word but still it is not a common place event. Neem tree, Azadirachta indica; is a medicinal plant. The trees contain many chemical compounds of which neem seed contain much azadirachtin. It possess medium to broad spectrum of action against insects selectively with low mammalian toxicity. Azadirachtin seems to be an "ecdysone blocker. The interferences of the azadirachtin is too insect’s specific against chemoreceptor and endocrine control systems, by which it affect insect growth, feeding and oviposition. Those are so different from the mammalian system that no toxic effects have been found even after application of gram amounts of azadirachtin per kilogram of body weight. When the neem seed oil is correctly formulated its efficiency increases many times, without any adverse effect on our ecology and environment.
A high frequency microcloning protocol for subsequent cryopreservation in Kae...iosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Production of Haploids Plants from Anther Culture of Musa Paradisiaca cv. ‘Pu...RSIS International
Haploid plants were regenerated from the anther callus of banana Musa paradisiaca (AB) cv. Puttabale. The highest frequency of callus induction (90%) was observed at the concentration of 3mg/l 2, 4-D . After 20 days of incubation organization of embyroids were organised from the callus mass. Interaction of 4mg/l BAP and 0.4 mg/l IAA provoked shoot growth of the embryoids and well organised roots were developed at the concentration of 0.6 mg/l NAA and the media was agumented with 0.2% activated charcoal. Flow cytometry study was carried out to analyse the DNA content of the regenerated haploid plants. The results of the investigation reported the efficient production of haploid plants from the anther culture.
ABSTRACT- Medicinal Plants have been practiced for hundreds of centuries by tribes all over the world. From the earliest times until the end of nineteenth century plants are still the common source of medicinal treatment yet. Using natural, plant-derived medicines that are “healthier” then prescription drugs derived from synthesized products is something that appeals to consumers. The medicinal plants are of great importance because there are utilized as medicines. Aim of this research work was to evaluate the antibacterial activity of Skimmia laureola plant against various patho-genic strains of bacteria. The hot and cold water extract of Skimmia laureola were used against four bacterial strains Escherichia coli,Bacillus subti-lus, Staphylococcusaureus and Proteus mirabilis in order to check the antibacterial activity of Skimmia laureola. Antibacterial activity was conducted by agar well diffusion method. The Skimmia laureola showed different level of antibacterial activity. The hot and cold water extract of Skimmia lau-reola showed antibacterial activity against the micro-organism but not too maximum. Keywords: Medicinal Plants, Skimmia Laureola, Antibacterial Activity.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Influence of Plant Growth Regulators on Somatic Embryogenesis Induction in Se...IJEABJ
Seriphidium herba-album (syn. Artemisia herba-alba) is a medicinal, aromatic, greenish-silver herb. It is used widely in folk medicine for treatment of diarrhea, abdominal cramps and in the healing of external wounds. It's also used for the treatment of diabetes mellitus, neurological disorders as epilepsy, Alzheimer’s disease, depression and jaundice. In this study we assessed the protocol for callus induction, maturation of somatic embryogenesis, frequency of germination and conversion into plantlets for leaf explants of Seriphidium herba-album using different concentrations of PGRs. Highest induction frequencies of embryogenic calli occurred after 35 days on MS medium supplemented with 1.5 mg L-1 2,4-D and 0.5 mg L-1 BAP. Optimum MS medium for higher frequency of matured somatic embryos was recorded using 5.0 mg L-1 BAP and 0.5 mg L-1 NAA and somatic embryos also induced young in vitro grown plantlets when cultured in the medium containing GA3 and kinetin. Hence, attempts to induce direct somatic embryogenesis have been achieved up to embryo regeneration and maturation.
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
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.
Burnt Weed Smoke Can Enhance Plant Growth A Proper Weed ManagementYogeshIJTSRD
Weeds are serious issue around the world causing crop yield reduction in agricultural fields. However, several studies proclaim the uses of weed plant species as plant growth enhancer because of their unique phytochemical composition present in smoke when pyrolysed. This idea has been inspired by the discovery of karrikins, a class of smoke elicitors that cues the seedling germination in several plant species. The present review is mainly aimed towards the application of weed derived smoke to regulate the plant growth in positive manner. Smoke water prepared from pyrolysed weed emerged out as more powerful in promoting the plant development of agriculturally and medicinally. The smoke technology can be one of the useful management strategies in future with cost effective and environmental friendly inputs. Shaiphali Saxena "Burnt Weed Smoke Can Enhance Plant Growth: A Proper Weed Management" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-3 , April 2021, URL: https://www.ijtsrd.com/papers/ijtsrd39954.pdf Paper URL: https://www.ijtsrd.com/biological-science/botany/39954/burnt-weed-smoke-can-enhance-plant-growth-a-proper-weed-management/shaiphali-saxena
Bio efficacy of pseudomonas fluorescens isolated from chickpea fields as plan...Shazia Shahzaman
Chickpea is an economically important food crop, which is subjected to infection by a host of fungal, viral and bacterial pathogens. Thirty isolates of Pseudomonas fluorescens were isolated from the rhizosphere of Chickpea fields. These were tested against F. oxysporum in dual culture method. Among these, four (Pf 1, Pf 3, Pf 5 and Pf
8) isolates were showed bright fluorescence under UV light were further tested. All the cultural and biochemical studies confirmed them to be P. fluorescens. The isolates also showed positive response for siderophore production and plant growth promoting activity on Chickpea cultivar Bital 98. Among these isolates Pf 3 and Pf 5 shown significant results by increasing root length and shoot length. Both the Pf 3 and Pf 5 isolates were found significantly superior than other isolates in increasing the shoot length (12.7 cm) and root length (24.5 cm) over control. The isolates Pf 3 was recorded high vigor index (3830) followed by Pf 5 (3648). The least vigor index was recorded by Pf 1 (2631).
Effect of Growth Retardants on Shoot and Root Development of Stevia(Steviareb...IOSRJAVS
Stevia rebaudianaBertoni is a natural sweetener herb, which is promising in food and pharmaceutical production. In Egypt, the gap between sugar production and consumption represents a serious problem, in order to close this gap; Stevia could be cultivated in the Egyptian environmental conditions. The objective of this study was to evaluate the effect of paclobutrazol(PBZ) and ethephon (ET) on shooting and rooting of stevia plant in vitroto improve the survival and growth of Stevia plantlets during acclimatization.The highest shoot number (48.0 shoots/explant) and the highest number of leaves (7.34) were recorded on MS medium containing 0.1mg/l PBZ. Whereas, the highest shoot length (7.36 cm) was obtained on MS medium without supplementation withPBZ or ET. The highest fresh and dry weights of shoots was recorded on MS medium containing 0.5 mg/l PBZ, which gave2.83 and 0.39 g, respectively. The highest number of roots (8.44 roots/shoot) was obtained with 0.1mg/l PBZ. Also, this study indicates that PBZ or ET increased chlorophyll a, chlorophyll b, carotenoids and carbohydrates content in the plant. PBZ and ET had significant effect on thesurvival of plants ex vitro, which recorded 85 and75%, respectively.
Callus Induction and Plantlet Regeneration in Orthosiphon aristatus (Blume) M...IOSR Journals
An efficient protocol was devised for rapid callus induction and plantlet regeneration from the leaves of Orthosiphon aristatus. For callus induction, auxins such as 2, 4-D, IAA, NAA alone and in combination with cytokinin BAP were used. The most effective medium for callus induction and shoot regeneration was M S medium fortified with 8mg/l BAP and 2mg/l NAA, on which multiple shoots were obtained after 15 days of callus induction. All the in vitro raised shoots with length of 3-5 cm were transferred to rooting medium supplemented with different concentrations of IBA. The best rooting response was observed on half strength M S liquid medium supplemented with 3mg/l IBA. The established plantlets obtained were subjected to hardening and acclimatisation by transferring to polycups containing sterile soil for 3-4 weeks and then to the field, where
85% survived to maturity
In vitro organogenesis protocol for Rauvolfia serpentina - an endangered medi...researchplantsciences
Rauvolfia serpentina commonly known as sarpagandha is a pharmacologically important medicinal plant containing numerous alkaloids with antibacterial, antidysentric and antidotal properties. The present study reports an efficient in vitro regeneration protocol by using nodal explants for this species. The sterilization technique was first standardized using ethyl alcohol, mercuric chloride and sodium hypochlorite with hot water and without hot water treatment. 100% aseptic culture was obtained when the explants were treated with hot water (at 500 C for 10 minutes) and 0.1% mercuric chloride. The aseptic cultures were inoculated in to culture medium with different concentrations of growth regulators. Higher explants response (78.33%) and higher multiple shoot formation from Rauvolfia serpentina nodal explants was observed in the medium supplemented with BAP (1mg/l) + KIN (1mg/l) + GA3 (0.5mg/l).
Article Citation:
Singh K and Dash M.
In vitro organogenesis protocol for Rauvolfia serpentina - an endangered medicinal plant.
Journal of Research in Plant Sciences (2012) 1(1): 083-088.
Full Text:
http://plantsciences.co.in/documents/PS0028.pdf
The ethanol extracts of Ficus asperifolia, Mormordica charantia, Anacardium
occidentals and Psidium guajava were evaluated sole and in treatment combinations at 25, 50 and
75mg ml-1 concentration levels against the mycelial growth of Macrophomina phaseolina of
Cowpea. The pathogen was cultured on plates containing botanicals amended Potato Dextrose
Agar (PDA) in three replicates while only ethanol treated PDA tested plates served the control
experiment. The radial growths were recorded at 4th, 6th and 8th day after inoculation. Data
obtained were analysed using the SAS software program version 9.2. The extract of Mormordica
charantia was the most effective in the botanical treatments alone. The most significant inhibition
of Macrophomina phaseolina were observed from the combined treatments of Ficus asperifolia,
Mormordica charantia and Anacardium occidentals (3.11 cm), followed by Mormordica
charantia and Psidium guajava (3.29 cm), then combination of four extracts; Ficus asperifolia,
Mormordica charantia, Anacardium occidentals and Psidium guajava (3.53 cm), then
Mormordica charantia and Anacardium occidentals (3.84 cm). Other treatments, either alone or in
combination produced significant result compared to the control experiment (6.94 cm). However,
the efficacy of botanicals increased with concentration and also significantly correlated with time
and reduction in mycelia extension of the pathogen. More so, variability in the antifungicidal
potentials of the botanicals on Macrophomina phaseolina ranges from 15.93% to 34.06%
according to Eigen proportions. The treatment combinations of; Ficus asperifolia, Mormordica
charantia and Anacardium occidentals at 75mg ml-1 concentration level produced the most
inhibitory effect against Macrophomina phaseolina in vitro. However, the untreated plates did not
show inhibitory effect on the mycelial growth of the pathogen. Therefore, combined treatments of
botanicals could be a potential source in the practice of plant disease control.
2. 2816 J. Med. Plants Res. diabetes, blood disorders, and throat infections and as a nerve tonic and works well as a sedative (Bussmann et al., 2013; Karato et al., 2006; Petrie and Peck, 2000). The efficacy of the oil is exploited in many major food, alcohol and beverage industries. Rose geranium is a native of dry rocky slopes of Cape Province in South Africa and was commercially cultivated in France, Belgium, Spain, Morocco, Madagascar, Egypt, Reunion Islands, Congo, China, India and the former USSR countries (Farooqi and Sreeram, 2001). The species was first introduced in Yercaud on the Shevroy Hills (1370 to 1525 m attitude) of Salem district of Tamil Nadu. Later, a number of scented and ornamental Pelargonium species were introduced into India, some of which got naturalized, escaped garden cultivation and are found growing wild on Shevroy, Kodaikanal, Nilgiri Hills of Tamil Nadu State and certain parts of Karnataka as it prefers high altitude areas with a milder climate (Rajeswara et al., 1999). Commercially, geranium is widely used for scenting soaps and high-grade perfumes due to the presence of low molecular weight aroma compounds (Verma et al., 2010; Farooqi and Sreeram, 2001). At one time, India was producing about 20 tonnes of geranium oil, with an average of nearly 1,400 hectares (Narayana et al., 1986). However, current Indian production has decreased to less than two tonnes per year due to significant reduction in the availability of cultivated area and ever changing climatic conditions. This has necessitated the application of modern biotechnological approaches/tools to increase the productivity to compete the requirements. The present study aims at formulating a protocol for increased rate of propagation and productivity of P. graveolens by in vitro culturing and genetic transformation.
MATERIALS AND METHODS
P. graveolens saplings were obtained from Horticulture Research Institute, Udagamandalam, The Nilgiris, Tamil Nadu. Soil from Ooty hills fertilized with “panchkavyam” was used to grow the saplings. The saplings were grown in shady region of the green house and watered once a day and maintained in the department nursery.
Callus induction
The leaf explants (Figure 1A) were initially washed thoroughly with tap water to remove the dust particles and soil residues, followed by rinsing in soap water for few minutes. Further, the explants were treated with 20% teepol and 10% bleach (Ala) for 10 min. Finally, the leaves were surface sterilized by agitating them in 0.1% HgCl2 for about 5 min to eliminate fungal and bacterial contaminants above and below the cell surface. The explants were rinsed thoroughly in sterile distilled water. The sterile leaf explants were cut into 5 mm long segments and plated on to Murashige and Skoog (1962) solid medium supplemented with appropriate combinations of nutrients and plant growth regulators (Table 1). The bottles were sealed and incubated in a growth chamber at 25°C under 16/8 h (light/dark) photoperiod for 25 to 30 days. Micropropagation Micropropagation was carried out using nodal bud explants in MS medium supplemented with auxins: cytokinins, 3% w/v sucrose and 0.7% w/v agar (Table 2). pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C for 30 min. The explants were incubated at 26 ± 1°C with 16/8 h light/dark cycles under white fluorescent lamps (3000 to 5000 lux). Shoots were regenerated from the callus by subjecting them in a cytokinin rich medium for 16/8 h light and dark photoperiod. Rhizogenesis Rhizogenesis was induced by sub culturing the in vitro regenerated shoots into rooting medium supplemented with higher concentra- tions of auxin and cytokinin and maintained under complete dark condition. Acclimatization The plantlets, with shoot and roots, were taken out from the culture medium and washed gently with double distilled water for removing all traces of medium from the surface. The plantlets were then transferred to small plastic cups containing sterile sand. The plastic cups were covered with sealed plastic vinyl bags to keep full humidity at 25 ± 2°C in light conditions (photon flux density at 25 μmol m-2 S-1, 16 h). The plantlets were moistened with water. As the plants grew vigorous, the bags were poked with chopsticks to allow air into the bags until the plants were self supported. The polythene bags were removed after fifteen to twenty days. The plantlets were later transferred to larger pots containing sterile sand and soil (1:1 ratio) and kept under shade in the green house.
Transformation studies
A 35 days old callus of P. graveolens was used for transformation using Agrobacterium tumifaciens LBA4404 containing a binary vector pTOK 233. Healthy compact callus from aseptically grown P. graveolens explant was transferred into a sterile petridish containing Whatmann filter paper. Callus was wounded by pricking with a sterile needle all over the surface. The explants were incubated for 48 h in MS medium to increase its meristematic activity. Overnight grown culture of A. tumefaciens strain with pTOK 233 in yeast extract peptone (YEP) with kanamycin was directly used for co-cultivation. Approximately 5 to 10 ml of Agrobacterium culture were poured into a sterile petridish, 3 to 10 callus explants were taken using sterile forceps and immersed and incubated for 10 min. After incubation, the explants were transferred to a petridish with sterile Whatmann No.1 filter paper. Explants were washed with MS containing 250 mg/L carbencillin to kill the Agrobacterium and again transferred to sterile petridish with Whatmann No. 1 paper. The washed explant was transferred to MS and incubated for 2 days in culture room. After 2 days, explants were aseptically transferred to selection medium (MS with hygromycin and 600 μl of 250 mg/L carbenicillin). The transformed callus carrying binary vector pTOK233 from A. tumefacie selection medium was cut into small sections. The explant was mixed with X-gluc solution and incubated for 2 to 12 h at 37°C in the dark. The tissue sections
3. Benazir et al. 2817 Table 1. Callus induction and proliferation from leaf explants of geranium.
S/No.
Treatment
Callus induction (After 16 days)
Callus proliferation (After 27 days)
Viable callus (After 45 days)
PGR
μM
1
2,4-D
10-60
0
0
0
2
NAA+
20
+ +
+ + +
Y
KIN
10
3
NAA+
20
+
+
N
ZEA
10
4
IAA+
20
+ + +
+ + + +
Y
KIN
10
5
IAA+
10
+ +
+ + +
Y
KIN
5
6
IAA+
20
+ +
+ + +
Y
BAP
10
7
IAA+
10
+ +
+ +
Y
BAP
5
8
IAB+
20
+ + + +
+ + + +
N
KIN
10
9
IBP+
20
+ + + +
+ + + +
Y
BAP
10
0: Explants turned brown and died, +: Only induction was there, no proliferation, ++: fair growth, callus was visible to naked eye, +++: good growth, callus covered the explants, ++++: excellent growth, prolific callus development.
were washed in ethanol. Tissues were stored in100 mM phosphate buffer and examined under dissection microscope.
RESULTS
Callus induction and regeneration
Callus was induced from the leaf explants cultured on MS medium supplemented with various combinations of indole-3-acetic acid (IAA), indole butyric acid (IBA), benzyl amino purine (BAP) and kinetin (Table 1). Curling and appearance of protuberances occurred in a time period of 4 to 6 days (Figure 1B). Callus induction was observed in 16 to 17 days of incubation in dark at 25°C. Proliferative callus was observed in 25 to 27 days of culturing (Figure 1C). Callus induction was initiated from the midrib and veins of the explants. Callus obtained in dark was white friable and compact. Callus when incubated in light turned green friable and nodular in 1 to 2 days. Pigmentation was observed in callus tissues incubated in light after about 8 to 10 days (Figure 1C). Maximum, proliferation was obtained in medium with combinations of MS with 20 μM IBA + 10 μM KIN, 20 μM IBA + 10 μM BAP and 20 μM IAA + 10 μM KIN. The callus was maintained by sub culturing in the same medium at an interval of 10 to 12 days. Proliferation of sub-cultured callus was observed within a time period of 2 to 3 days (Table 1, Figure 1C and D). Shoots were emerged as tiny pale green even globules from 3 to 5 days old proliferative callus tissue grown under 16/8 h light/dark photoperiod at 25°C (Table 2, Figure 1D and E). The callus tissues were found responsive to higher concentrations of cytokinin. The shoot primordial developed had white petiole carrying un- incised leaf, which later showed incisions and lobes.
They were light green during the time of incubation and turned dark green as they grew, then later on acquired red pigmentation. Shoots without pigmentation were also observed in callus tissues grown in complete dark condition. Maximum proliferation and multiplication of
4. 2818 J. Med. Plants Res. Table 2. In vitro regeneration of shoots and roots from leaf derived calli of geranium.
PGR’s
Shoot regeneration
(16 h light/8 h dark)
Rhizogenesis (16 h light/8 h dark)
PGR
μM
5 d
15 d
30 d
5 d
15 d
30d
BAP
20
+ +
++ +
-
← Shooting medium
KIN
20
+ +
+ +
-
BAP+
20
+ + +
+ + +
+ + +
IAA
5
KIN +
20
+ +
+ + +
+ ++
IAA
25
KIN +
10
+ +
+ + +
+ +
IAA
10
KIN +
10
+ +
+ + +
+ +
IBA
10
IBA +
20
← Rooting medium
-
+
+ + +
KIN
10
IBA +
20
-
+
+++
BAP
10
IAA+
20
-
+
++++
BAP
5
IBA +
20
-
+
++++
BAP
5
++: 1-2 shootlets, +++: few shootlets, ++++: numerous shootlets, +: induction of root, visible to naked eye, +++: proliferation, +++: maximum proliferation.
shoots was observed in hormone combinations of 20 μM BAP + 5 μM IAA, 20 μM KIN + 5 μM IAA. After multiplication had started, the shoots showed elongation of petiole, which was initially white, then turned green in color. Complete proliferation was obtained after 15 to 16 days (Figure 1E and F). Different stages of shoot buds, from induction to whole bud formation was observed. It appeared as small protuberance that gave rise to nodules of globular and bent shape. These nodules on further differentiation showed dome shaped structure with apical incision, which were deepened further. The shoot buds which differentiated from the callus were meristematic in nature (Figures 1G and H). Micropropagation
The nodal bud explants maintained in BAP and KIN supplemented medium initiated shoot development by 3 to 4 days of inoculation. Prominent shoot multiplication was observed around 15 to 20 days of culturing in shooting medium. The shoot development was rapid and covered the culture tube in 28 days. Numerous shootlets were developed when callus tissue was sub cultured in MS medium with 10 μM BAP + 5 μM IAA and 20 μM KIN + 5 μM IAA under continuous white light. All the shootlets had green petiole with lobed leaves and glandular hairs (Figure 1I). Rhizogenesis
Roots were induced from the in vitro regenerated shoots grown in medium supplemented with higher concen- tration of auxin:cytokinin growth hormones (Table 2). Small hairy fibrous roots were developed from the shoot base after 20 to 25 days of culturing. The number of rhizome developed was observed maximum in medium supplemented with 20 μM IBA + 5 μM BAP (Figure 1J). Regenerated plantlets were acclimatized under controlled in vivo condition and transferred to pots and maintained (Table 2, Figure 1K).
5. Benazir et al. 2819 Figure 1. In vitro regeneration of P. graveolens
Transformation
An effficient protocol for transfer of genomic DNA into P. graveolens using Agrobacterium mediated transformation was developed. After 12 h incubation at 37°C in dark, transformed calli showed transient blue coloration while un-transformed calli showed no sign of colorations. Transformed calli harbored the plasmid pTOK 233 which contain the β-glucuronidase gene, which metabolized X- gluc and gave characteristic blue color (Figure 1L and M).
DISCUSSION
The present study aims at formulating a protocol for increased rate of propagation and productivity of P. graveolens by in vitro culturing and genetic transfor- mation. The geranium oil is an indispensable essential oil in aromatherapy and perfume industry. Manipulation at molecular level to develop stable transgenic plant with increased oil production is a substitute to save the biomass. The genetic transformation attempted as a part
6. 2820 J. Med. Plants Res. of this study, thus finds a significant application in increasing the production of essential oil and fragrance quality of this aromatic plant. MS medium supplemented with various levels of 2,4-D was chosen as primary medium for callus induction but unlike the studies reported in P. graveolens (Sreedhar, 1999), the plant showed poor response and no sign of growth. This may be due to improper modulation of exogenous auxin and endogenous auxin content that would prove antagonistic to callus induction. This implies that the initiation of a proliferating culture from explants, involves profound change in the development state of the tissue and relates to the alterations in the basic architecture of cells or tissues resulting in the activation of quiescent cells (Irfan, 2001). Curling of tissue and protuberances were formed that was followed by the appearance of little irregular cellular masses that were observed around the pricked end, which may be due to exogenous substances that ooze out of the injured tissue at cut end and stimulates cell division (Kumar, 1992). The callus induction was observed from the midrib as it is rich in meristamatic tissue, and meristematic tissue have high rate of growth as they are physiologically active (Razdan, 2003). Substantial callus was formed in a time period of 25 to 27 days, as usually it takes 4 weeks for complete callus formation (Kumar, 1992). The texture of callus observed was friable and compact. Texture varies according to explant and species used (Kumar, 1992). Callus was colorless in dark and acquired green color when exposed to light, which is probably due to photo stimulation of chloroplast and development of chloroplastids in the callus tissue. Some callus tissues turned purple in color which may be due to accumulation of anthocyanins (Kumar, 1992). Maximum proliferation was obtained in medium with hormone combination MS with 20 μM IBA + 10 μM KIN, 20 μM IBA + 10 μM BAP and 20 μM IAA +10 μM KIN. This shows that a good modulation of auxin and cytokinin is required for formation of callus from geranium leaves unlike auxins alone as reported in many cases (Razdan, 2003; Batra, 2001) (Figure 1C and D). Shoot induction occurred when a photo period of 16/8 h light/dark was provided as geranium is reported to be highly sensitive to light (Sreedhar, 1999) and regeneration was delayed when exposed to light alone (Sreedhar, 1999). The differential coloration of shootlets was also observed. Albinos were formed in the dark due to lack of chloroplastids while in light, the chloroplastid differentiation was induced and procured green color (Kumar, 1992). The pink coloration in leaf was obtained, which can be the manifestation of somaclonal variation (Brown and Charlwood, 1986) or deposition of anthocyanin pigments (Razdan, 2003). Caulogenesis occurs in blue light or low intensity white light or in the dark with sudden exposure to light (Kumar,
1992). Shootlets grew into the medium and shows its sensitivity to longer period of light conditions (Sreedhar, 1999). Maximum shoot proliferation was obtained in hormone combinations MS with 20 μM BAP + 5 μM IAA and 20 μM KIN + 5 μM IAA. This shows that the plant requires large amounts of cytokinin with auxin supplement for shoot proliferation (Saxena et al., 2001) (Figure 1D to F). Multiplication of the shoots were observed, when the callus with shoot buds was sub cultured to MS supplemented with 20 μM BAP + 5 μM IAA, 20 μM kin + 5 μM IAA. Saxena (2002) in her work on in vitro procedure for micropropagation had that found MS 0.5 mg/L BAP + 0.1 mg/L NAA exhibited regeneration of maximum number of shoots. Whereas, Sreedhar (1999) had reported that the maximum number of shoots for in vitro propagation of geranium was observed in medium containing BAP and IAA at 1 mg/L. Different stages of shoot buds from induction to whole bud formation were observed in simple light mode of phase contrast microscope. The stages like nodular, dome shape bend shape, dome shapes with apical incision were observed. The protuberances seem to differentiate from epidermal cells and have become meristamatic. These stages are confirmed to be stages in shoot bud formation, as there are reports of histological analysis of shoot differentiation in Chloroxylon swietenia. Like in geranium, the microscopic examination of the Chloroxylon callus from hypocotyl region revealed that mitotic division actually initiated in pericyclic region, giving rise to a mass of compactly arranged cells that contained dense cytoplasm and prominent nuclei. In pericyclic region, due to continuous proliferation of cells, a pressure was developed from the radial growth of the cells, which led to the disruption and degeneration of the cortex triggering some of the newly formed cells to exhibit re-differentiation. These cells generally occurred in nests or nodules and could be appropriately referred to as „growth centres‟ or „meristermoids‟ that further differentiated to dome shaped structure. Oval and elongated callus cell with thick wall were also viewed in microscopical examination. This corresponds with the differentiation of callus tissues at the time of initiation and further growth shows mixed population of small, more rounded, oval and few elongated cells with dense cytoplasm. In geranium callus, the roots formed were small, hairy, and fibrous and resembled that of field grown plant. The callus tissue developed slight yellowish brown pigmen- tation during rhizogenesis, (Kumar, 1992). The hormone combination MS 20 μM IBA + 10 μM BAP resulted in the highest amount of rhizogenesis. Similar observation was reported by Razdan (2003), that development of roots under light and dark suggest that the rhizogenesis in geranium is insensitive to photoperiods and high level
7. of auxin favours the multiplication. Regeneration occurs by complete organogenesis, usually initiation of shoot buds in calli may precede rhizogenesis or sometimes it succeeds rhizogenesis as reported by other studies (Kumar, 1992). The latter case was observed in the gera- nium callus. The callus for rhizogenesis was incubated in the same medium (MS + 20 μM IBA + 5 μM BAP), which gave rise to shootlets after 45 days of incubation. Nodal buds inoculated in MS medium supplemented with 20 μM IAA + 10 μM KIN gave rise to first, a single shoot, then to multiple shoots, unlike the combination given by Saxena (2001) on geranium which was 8 mg/L KIN and 1 mg/L NAA. It took 15 to 20 days for multiplication, unlike easy breaking of dormancy of shoot bud; this may be due to usage of more auxin concentration than cytokinin. The appearance of milky white callus can also be attributed due to the auxin and cytokinin combination with more of auxin that indeed promotes callus induction (Razdan, 2003). The transformed calli pieces showed transient blue coloration. The callus was highly proliferated, and friable pre- incubation requirement was only 2 days. Pre-culture is crucial because host cell division is required for successful Agrobacterium transformation (Binns and Thomashow, 1988), it is not surprising that pre-culture in a high auxin medium often enhances transformation rate (Mathis and Hinchee, 1994; Sangwan et al., 1992). In addition, because there was no effect of pre-culture or transient gb-glucuronidase (GUS) expression, it is likely that plants benefit from pre-culture by increasing the competence of cells for DNA incorporation into chromosome and/or de-differentiate into callus rather than enhancing DNA transfer cells. Callus was chosen, as it was highly meristematic compared to other explants. As per the standardized protocol for Agrobacterium transformation in Nicotiana tobaccum, in the present study, the infection time and incubation time for co- cultivation is respectively 8 to 10 min and 2 days. The same time intervals were given for geranium calli treatment and to prevent the bacterial overgrowth, selection on hygromycin and carbenicillin containing medium was carried out for 2 days, which prevented the bacterial growth. Selection was carried out in dark as these antibiotics are light sensitive (Meilan et al., 2002). The transient expression is a test for plasmid transformation into calli. The plasmid pTOK 233 harbors β-glucuronidase gene that is capable of metabolising X- gluc and gives blue color. The transient expression even though is a positive confirmatory test for transformation (Jefferson, 1987) can sometimes get mislead due to incorporation of bacteria into callus which has not been selected properly on antibiotic medium (Han et al., 2000). Thus we have developed a rapid protocol for the transformation of genomic DNA into P. graveolens using A. tumefaciens.
Benazir et al. 2821
Conclusion
The present study aimed to establishing an efficient protocol for in vitro regeneration and Agrobacterium mediated transformation of P. graveolens as an alternative method for vegetative propagation. The results suggest better methods of propagation and efficient utilization of the in vitro protocols for the mass multiplication of this commercially important crop. Further, the transformation study provides a primary step for rapid gene transfer towards the improvement of the plant for oil content and fragrance at the molecular level. REFERENCES Batra A (2001). Micropropagation In: Fundamentals of plant biotechnology, Capital Publishing Company, New Delhi, Calcutta. pp. 18-23. Binns AN, Thomashow MF (1988). Cell biology of Agrobacterium infection and transformation of plants. Ann. Rev. Microbiol. 42:575- 606. Brian AK, Brian EW, Ingram M, Brenda C (2010). Geranium Leaf Tissue Nutrient Sufficiency Ranges by Chronological Age. J. Plant Nutr. 33(3):339-350. Brown JT, Charlwood BV (1986). The accumulation of essential oils by tissue cultures of Pelargonium fragrans (Wild). Fed. Eur. Biochem. Soc. 204:117-120. Bussmann RW, Paniagua ZN, Chamorro MR, Moreira NM, del Rosario CNML, Olivera J (2013). Peril in the market-classification and dosage of species used as anti-diabetics in Lima, Peru. J. Ethnobiol. Ethnomed. 30:9-37. Dormon HJ, Deans SG (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88(2):308-316. Farooqi AA, Sreeram BS (2001). Geranium. In: Cultivations of medicinal and aromatic crop. pp. 356-362. Han KH, Meilan C, Ma C, Strauss SH (2000). An Agrobacterium tumefaciens protocol effective on a variety of cotton wood hybrids (genus Poplus). Plant Cell Rep. 19:315-320. Irfan KA (2001). Role of Biotechnology in Medicinal and Aromatric Plants. Razdan, MK. Introduction to Plant Tissue culture. p. 16 Jefferson RA, Kavanagh TA, Bevan MW (1987). GUS Fusions: β- glucosidase as sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901-3907. Jeon JH, Kim HW, Kim MG, Lee HS (2008). Mite control activities of active constituents isolated from Pelargonium graveolens against house dust mite. J. Microbiol. Biotechnol. 18(10):1666-1671. Karato M, Yamaguchi K, Takei S, Kino T, Yazawa K (2006). Inhibitory effects of pasuchaca (Geranium dielsianum) extract on alpha- glucosidase in mouse. Biosci. Biotech. Biochem. 9(6):1482-1484. Kumar De K (1992). Callus culture, Cytodifferentiation, Organogenesis. In: Plant tissue cultures. pp. 35-78. Mathis NL, Hinchee MAW (1994). Agrobacterium inoculation techniques for plant tissues. In: Gelvin SB, Schilperoort, RA (eds) Plant molecular biology manual. Kluwer Academic Publisher, Boston. pp.1-9. Matthews AJ (1995). Geranium leaves for cracked nipples. Aust. J. Hosp. Pharm. 25:538-539. Meilan R, Han KH, Ma C, Di Fazio SP, Eaton JA, Hoien EA, Stanton BJ, Crockett RP, Taylor ML, James RR, Skinner JS, Jouanin L, Pilate G, Stauss SH (2002). The CP4 transgene provides high levels of tolerance to Roundupw herbicide in field-grown hybrid poplars. Can. J. For. Res. 32:967-976. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15:473-497.
8. 2822 J. Med. Plants Res. Narayana MR, Prakash R, Rajeswara RBR, Sastry KP (1986). Geranium cultivation in India: Potentials and prospects. PAFAI. 4:25- 29. Ooshiro A, Hiradate S, Kawano S, Takushi T, Fujii Y, Natsume M, Abe H (2009). Identification and activity of ethyl gallate as an antimicrobial compound produced by Geranium carolinianum. Weed Biol. Manage. 9(2):169-172 Petrie KA, Peck MR (2000). Alternative medicine in maternity care. Prim. Care. 27:117-136. Rajeswara RBR, Bhattacharya AK, Kaul PN (1999). Rose Scented Geranium (Pelargonium graveolens) cultivation and technology for Andhra Pradesh. Indian Perfumer. 43(1):2-8. Rajeswara RBR, Bhattacharya AK, Kaul PN, Ramesh S (1992). The essential oil profiles of Rose Scented Geranium (Pelargonium Sp.) biomass dried prior to distillation. Indian Perfumer. 36(4):238-240. Razdan MK (2003). Cellular Totipotency, In: Introduction to plant tissue culture second edition : Oxford IBH Publishing Company Limited, New Delhi. pp. 48-49. Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan Norreel B (1992). Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta. 188:439-456. Saxena G, Banerjee S, Rahman L, Mallavarpu GR, Sharma S, Kumar S (2001). An efficient, in vitro procedure for micropropagtion and generation of somaclones of Rose scented Geranium. Pubmed. pp. 1081-1086.
Sreedhar D (1999). Tissue culture of Pelargonium graveolens (Geranium). In: Role of Biotechnology in Medicinal and Aromatic Plants. Vol.2. Eds:Irfan. A.Khan, Atiya Khanum . Ukaaz Publications. pp. 177-184. Tabanca N, Wang M, Avonto C, Chittiboyina AG, Parcher JF, Carroll JF, Kramer M, Khan IA (2013). Bioactivity-Guided Investigation of Geranium Essential Oils as Natural Tick Repellents. J. Agric. Food Chem. 25:1-7.
Verma RS, Verma RK, Yadav AK, Amit C (2010). Changes in the essential oil composition of rose-scented geranium (Pelargonium graveolens L Herit.ex. Ait.) due to date of transplanting under hill conditions of Uttarakhand. Indian J. Nat. Prod. Resourc. 1(3):367- 370.