A high frequency microcloning protocol for subsequent cryopreservation in Kaempferia galanga L.: An endangered, over exploited medicinal plant in pharmaceutics
This document describes a high frequency micropropagation protocol for Kaempferia galanga, an endangered medicinal plant. Key findings:
- Shoot cultures were established from rhizome explants on MS medium with BA and multiplied through subculturing. Maximum shoot multiplication (10.6 shoots/explant) was achieved on MS with 4 mg/L BA, 1 mg/L kinetin, and 1 mg/L NAA.
- Repeated subculturing every 4 weeks increased shoot multiplication 3-4x, allowing production of up to 30 shoots/explant within 12 weeks.
- Shoots elongated and rooted simultaneously without needing separate media, reducing production time versus previous protocols.
-
Organogenic Regeneration of an Elite Cultivar of Chinese Jujube (Zizyphus juj...Agriculture Journal IJOEAR
Abstract— An efficient and relatively simple regeneration system was developed for an elite cultivar of Chinese Jujube, a perennial tree, by culturing young twig segments as explants from 8-15 year old trees. The twig segments were disinfected by submerging them in 1% sodium hypochlorite (NaOCl) for 15 min with 3 min vacuum. Calli developed from both ends of the twig segments on half-strength MS medium supplemented with sucrose and BA or BA and NAA in combination. The frequency of shoot formation from calli was higher than 80% when the explants were placed on the half - strength MS medium supplemented with BA (2.581 μM) and NAA (2.685 μM). Roots were produced from adventitious buds for 90% of the regenerated shoots when they were placed on the MS medium supplemented with 4.920 μM IBA and 5.708 μM IAA. After transplanting to soil, 82% of the regenerated seedlings survived when they were covered with glass containers to maintain humidity. The results suggest that Chinese jujube can be reproduced and multiplied using organogenesis with the appropriate explant and culture medium.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
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.
Micropropagation of Santalum Album L. Sandalwoodijtsrd
An efficient plant regeneration protocol was developed for Santalum album L. Santalaceae , an economically important species. Plant regeneration was achieved using nodal explants and leaf disc on Murashige and Skoog MS medium for direct shoot regeneration. Effect of Plant Growth Regulators PGR like 6 Benzyl Adenine BA , Kinetin KN and 2 Isopentenyl adenine 2 iP on shoot initiation 2 Isopentenyl adenine and Gibberellic acid GA3 for shoot elongation and multiple shoot formation and Indole 3 Butyric Acid IBA and a Naphthalene Acetic Acid NAA for rooting was studied. Among the explants tested for shoot induction, nodal segments proved good results. The best treatment for obtaining shoot induction was 3.0mg L BAP and for rooting 1 mg L of IBA was found to be the best treatment combination for maximum sprouting of shoot and rooting. After six the rooted plantlets were transferred for hardening, 20 of plantlets survived and resumed growth in the mixture of soil, vermiculite and sand 1 1 1 . S. Aghi Zion Inbakani | S. Sathishkumar | Bakan Jagdish Sudhakar "Micropropagation of Santalum Album L. (Sandalwood)" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-4 , June 2021, URL: https://www.ijtsrd.compapers/ijtsrd43698.pdf Paper URL: https://www.ijtsrd.combiological-science/biotechnology/43698/micropropagation-of-santalum-album-l-sandalwood/s-aghi-zion-inbakani
Organogenic Regeneration of an Elite Cultivar of Chinese Jujube (Zizyphus juj...Agriculture Journal IJOEAR
Abstract— An efficient and relatively simple regeneration system was developed for an elite cultivar of Chinese Jujube, a perennial tree, by culturing young twig segments as explants from 8-15 year old trees. The twig segments were disinfected by submerging them in 1% sodium hypochlorite (NaOCl) for 15 min with 3 min vacuum. Calli developed from both ends of the twig segments on half-strength MS medium supplemented with sucrose and BA or BA and NAA in combination. The frequency of shoot formation from calli was higher than 80% when the explants were placed on the half - strength MS medium supplemented with BA (2.581 μM) and NAA (2.685 μM). Roots were produced from adventitious buds for 90% of the regenerated shoots when they were placed on the MS medium supplemented with 4.920 μM IBA and 5.708 μM IAA. After transplanting to soil, 82% of the regenerated seedlings survived when they were covered with glass containers to maintain humidity. The results suggest that Chinese jujube can be reproduced and multiplied using organogenesis with the appropriate explant and culture medium.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
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.
Micropropagation of Santalum Album L. Sandalwoodijtsrd
An efficient plant regeneration protocol was developed for Santalum album L. Santalaceae , an economically important species. Plant regeneration was achieved using nodal explants and leaf disc on Murashige and Skoog MS medium for direct shoot regeneration. Effect of Plant Growth Regulators PGR like 6 Benzyl Adenine BA , Kinetin KN and 2 Isopentenyl adenine 2 iP on shoot initiation 2 Isopentenyl adenine and Gibberellic acid GA3 for shoot elongation and multiple shoot formation and Indole 3 Butyric Acid IBA and a Naphthalene Acetic Acid NAA for rooting was studied. Among the explants tested for shoot induction, nodal segments proved good results. The best treatment for obtaining shoot induction was 3.0mg L BAP and for rooting 1 mg L of IBA was found to be the best treatment combination for maximum sprouting of shoot and rooting. After six the rooted plantlets were transferred for hardening, 20 of plantlets survived and resumed growth in the mixture of soil, vermiculite and sand 1 1 1 . S. Aghi Zion Inbakani | S. Sathishkumar | Bakan Jagdish Sudhakar "Micropropagation of Santalum Album L. (Sandalwood)" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-4 , June 2021, URL: https://www.ijtsrd.compapers/ijtsrd43698.pdf Paper URL: https://www.ijtsrd.combiological-science/biotechnology/43698/micropropagation-of-santalum-album-l-sandalwood/s-aghi-zion-inbakani
With the advancement of biotechnology, Genetic engineering also become an important tool. Transgenic crops are the crops which are produced through genetic engineering by altering desirable traits into plant genome.
Biotechnology in capsule form. Plant biotechnology . Industry related information. Latest info. Different aspects of plant biotechnology. Cell biology. industrial biotechnology.
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.
Vesicular Arbuscular Mycorrhizal Fungal status on some medicinal plants of Go...inventionjournals
Medicinal plants are important for our existence that supplies us many components for drug formulation. In nature the plant of particular kind invades with so many microorganisms. Among them one beneficial one is Va-mycorrhizal fungi. It helps in various ways to promote growth and yield of biomass better in natural habitats. So, to promote growth in garden or manmade environment application of VA-fungi as biofertilizer is beneficial. In this study 41 medicinal plants have been studied and application of VAM fungi inocula on Catharanthus roseus (L.) G. Don. have been done. Monsoon showed highest colonization percentage followed by winter and summer where as spore density showed highest during winter followed by summer and monsoon.
Evaluation of fungicides for their efficacy against seed-borne fungi of Groun...ICRISAT
Groundnut germplasm exchange has played a significant role in crop improvement programmes by providing wide genetic diversity world over. However, there is an inherent risk of introduction of new exotic pathogens or new/more virulent races of a pathogen into new areas. The basic principle of plant quarantine is to regulate the movement of seed material to mitigate the associated pest risk. Salvaging, a process of disinfecting infected seed, is an important quarantine strategy to prevent the introduction of new pathogens or virulent races into new areas.
4 ijfaf nov-2017-2-allelopathic effect of eucalyptusAI Publications
Laboratory and greenhouse experiments were carried out at the Faculty of Agricultural Sciences, University of Gezira, Sudan in season 2014/15 to study the allelopathic effects of Eucalyptus (Eucalyptus camaldulensis Dehnh) leaf on seed germination and seedling growth of some poaceous crops. Laboratory experiments were conducted to study the allelopathic effects of leaf aqueous extract of Eucalyptus on seed germination of sorghum (Sorghum bicolor [L.] Moench), millet (Pennisetum glaucum [L.] R. Br.), maize (Zea mays L.) and wheat (Triticum vulgare L.). Six concentrations (0, 20, 40, 60, 80 and 100%) of the leaf aqueous extract of Eucalyptus were prepared from the stock solution (50 g / l). Treatments, for each crop, were arranged in completely randomized design with four replicates. The seeds were examined for germination at three days after initial germination. Greenhouse experiments were conducted to study the allelopathic effects of Eucalyptus leaf powder on seedling growth of the same crops. The leaf powder of Eucalyptus was incorporated into the soil at rate of 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% on w/w bases in pots. Treatments, for each crop, were arranged in completely randomized design with four replicates. The experiments were terminated at 30 days after sowing and the plant height, number of leaves and root length of crop seedlings were measured as well as plant fresh and dry weight. Data were subjected to analysis of variance procedure. Means were separated for significance using Duncan`s Multiple Range Test at p 0.5. The results showed that the leaf aqueous extract of Eucalyptus significantly reduced seed germination of the tested poaceous crops and there was direct negative relationship between concentration and germination. Also, the results showed that incorporating leaf powder of Eucalyptus into the soil significantly decreased plant height, number of leaves and root length of crop seedlings as well as seedling fresh and dry weight. In addition, the reduction in seedling growth was increased as the leaf powder increased in the soil. Based on results supported by different studies, it was concluded that Eucalyptus has allelopathic effects on seed germination and seedling growth of the poaceous crops.
With the advancement of biotechnology, Genetic engineering also become an important tool. Transgenic crops are the crops which are produced through genetic engineering by altering desirable traits into plant genome.
Biotechnology in capsule form. Plant biotechnology . Industry related information. Latest info. Different aspects of plant biotechnology. Cell biology. industrial biotechnology.
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.
Vesicular Arbuscular Mycorrhizal Fungal status on some medicinal plants of Go...inventionjournals
Medicinal plants are important for our existence that supplies us many components for drug formulation. In nature the plant of particular kind invades with so many microorganisms. Among them one beneficial one is Va-mycorrhizal fungi. It helps in various ways to promote growth and yield of biomass better in natural habitats. So, to promote growth in garden or manmade environment application of VA-fungi as biofertilizer is beneficial. In this study 41 medicinal plants have been studied and application of VAM fungi inocula on Catharanthus roseus (L.) G. Don. have been done. Monsoon showed highest colonization percentage followed by winter and summer where as spore density showed highest during winter followed by summer and monsoon.
Evaluation of fungicides for their efficacy against seed-borne fungi of Groun...ICRISAT
Groundnut germplasm exchange has played a significant role in crop improvement programmes by providing wide genetic diversity world over. However, there is an inherent risk of introduction of new exotic pathogens or new/more virulent races of a pathogen into new areas. The basic principle of plant quarantine is to regulate the movement of seed material to mitigate the associated pest risk. Salvaging, a process of disinfecting infected seed, is an important quarantine strategy to prevent the introduction of new pathogens or virulent races into new areas.
4 ijfaf nov-2017-2-allelopathic effect of eucalyptusAI Publications
Laboratory and greenhouse experiments were carried out at the Faculty of Agricultural Sciences, University of Gezira, Sudan in season 2014/15 to study the allelopathic effects of Eucalyptus (Eucalyptus camaldulensis Dehnh) leaf on seed germination and seedling growth of some poaceous crops. Laboratory experiments were conducted to study the allelopathic effects of leaf aqueous extract of Eucalyptus on seed germination of sorghum (Sorghum bicolor [L.] Moench), millet (Pennisetum glaucum [L.] R. Br.), maize (Zea mays L.) and wheat (Triticum vulgare L.). Six concentrations (0, 20, 40, 60, 80 and 100%) of the leaf aqueous extract of Eucalyptus were prepared from the stock solution (50 g / l). Treatments, for each crop, were arranged in completely randomized design with four replicates. The seeds were examined for germination at three days after initial germination. Greenhouse experiments were conducted to study the allelopathic effects of Eucalyptus leaf powder on seedling growth of the same crops. The leaf powder of Eucalyptus was incorporated into the soil at rate of 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% on w/w bases in pots. Treatments, for each crop, were arranged in completely randomized design with four replicates. The experiments were terminated at 30 days after sowing and the plant height, number of leaves and root length of crop seedlings were measured as well as plant fresh and dry weight. Data were subjected to analysis of variance procedure. Means were separated for significance using Duncan`s Multiple Range Test at p 0.5. The results showed that the leaf aqueous extract of Eucalyptus significantly reduced seed germination of the tested poaceous crops and there was direct negative relationship between concentration and germination. Also, the results showed that incorporating leaf powder of Eucalyptus into the soil significantly decreased plant height, number of leaves and root length of crop seedlings as well as seedling fresh and dry weight. In addition, the reduction in seedling growth was increased as the leaf powder increased in the soil. Based on results supported by different studies, it was concluded that Eucalyptus has allelopathic effects on seed germination and seedling growth of the poaceous crops.
Similar to A high frequency microcloning protocol for subsequent cryopreservation in Kaempferia galanga L.: An endangered, over exploited medicinal plant in pharmaceutics
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In vitro regeneration and transformation studies on Pelargonium graveolens (g...Fathima Benazir
Development of improved protocols for the regeneration of Pelargonium graveolens is important for their commercial utilization. Callus was induced from leaf explants on Murashige and Skoog (MS) medium supplemented with auxins and combinations of auxins and cytokinins. Micropropagation of nodal bud explants was achieved using medium supplemented with cytokinins. The regenerated explants were acclimatized and the transformation potential of geranium was demonstrated using Agrobacterium tumefaciens. Maximum proliferation was obtained in medium supplemented with combinations of 20 μM indole butyric acid (IBA) + 10 μM kinetin (KIN), 20 μM IBA + 10 μM benzyl amino purine (BAP) and 20 μM indole-3-acetic acid (IAA) + 10 μM KIN. Plantlets were regenerated when callus was given a photoperiod of 16 h light and 8 h dark conditions at 25°C. After being transferred to greenhouse conditions, the plantlets were successfully acclimatized and transferred to the field condition. The study has established an efficient protocol for in vitro regeneration and transformation of P. graveolens as an alternative method for vegetative propagation.
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
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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,
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combination produced significant result compared to the control experiment (6.94 cm). However,
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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.
Assessment of Endophytic Fungal Flora Responsible for Plant Growth Promotion...Sryahwa Publications
The present paper discusses the highest colonization of fungal endophytes as Alternaria speciesin comparison with Colletotrichumspecies and Fusarium species in all three plants Pongamia pinnata, Securinega leucopyrus and Rhus mysorensis. These endophytic fungi protect these plants from various
environmental factors such as temperature, moisture and other environmental factors.
Plant tissue culture is used widely in the plant since , forestry and in horticulture .
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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
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.
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.
Mutation Induction for Improvement of Banana (Musa Spp). "Berangan Cv. Intan-...paperpublications3
Abstract: In vitro mutation induction by using gamma ray at 20, 30, 40 and 60Gy was used to generate variability in triploid banana "Berangan cv. Intan (AAA), so as to provide the opportunity to select plants with desirable characters such as early fruiting and short stature. Mutation frequency increased with increased dosage whereas, survival and capacity to regenerate decreased with increased doses. Time to initiation varied from 4-8 weeks for gamma-irradiated materials compared to 2-3 weeks in the control. It appeared that the higher the dose, the longer it took for shoot initiation. The exposure of shoot-tip meristem pieces to radiation doses produced wide variation in growth and morphogenetic performance. Mutagenic treatments induced 2 to 3- fold increases in variability in both quantitative and qualitative traits at different stages, in vitro; at nursery and field. For the field-grown plants; the proportion varied from 2.9% for the control plants to 16.8% for 60 Gy and 20.1% for 40 Gy-treatments, while for treatments at 20 and 30 Gy variations was at 18% and 19.2%, respectively. The frequency of variants was highest in 40 Gy followed by 30 Gy and 20 Gy, while it was very low in 60 Gy except for plant stature (dwarfism or stunted growth). Earliness to flowering variants were recorded at 20, 30 and 40 Gy at low frequencies (0.6%, 0.7% and 1.7%) respectively, while none was observed for 60 Gy treatments. As in vitro mutation induction could create genetic variability as well as many undesirable variants, it is highly desirable to integrate in vitro mutation with a selection system that can screen for large mutagen treated population. The useful variants recorded for earliness to flowering were selected for 30 and 40 Gy treatments. 40 Gy showed high frequency in earliness as compared with 20 and 30 Gy., which came to flowering as early as 6 – 6.5 months compared to 7-8 months for control. The useful dwarf, which considered as desirable traits, showed a balance of height and girth.
Mutation Induction for Improvement of Banana (Musa Spp.) Berangan Cv. Intan-AAApaperpublications3
Abstract: In vitro mutation induction by using gamma ray at 20, 30, 40 and 60Gy was used to generate variability in triploid banana "Berangan cv. Intan (AAA), so as to provide the opportunity to select plants with desirable characters such as early fruiting and short stature. Mutation frequency increased with increased dosage whereas, survival and capacity to regenerate decreased with increased doses. Time to initiation varied from 4-8 weeks for gamma-irradiated materials compared to 2-3 weeks in the control. It appeared that the higher the dose, the longer it took for shoot initiation. The exposure of shoot-tip meristem pieces to radiation doses produced wide variation in growth and morphogenetic performance. Mutagenic treatments induced 2 to 3- fold increases in variability in both quantitative and qualitative traits at different stages, in vitro; at nursery and field. For the field-grown plants; the proportion varied from 2.9% for the control plants to 16.8% for 60 Gy and 20.1% for 40 Gy-treatments, while for treatments at 20 and 30 Gy variations was at 18% and 19.2%, respectively. The frequency of variants was highest in 40 Gy followed by 30 Gy and 20 Gy, while it was very low in 60 Gy except for plant stature (dwarfism or stunted growth). Earliness to flowering variants were recorded at 20, 30 and 40 Gy at low frequencies (0.6%, 0.7% and 1.7%) respectively, while none was observed for 60 Gy treatments. As in vitro mutation induction could create genetic variability as well as many undesirable variants, it is highly desirable to integrate in vitro mutation with a selection system that can screen for large mutagen treated population. The useful variants recorded for earliness to flowering were selected for 30 and 40 Gy treatments. 40 Gy showed high frequency in earliness as compared with 20 and 30 Gy., which came to flowering as early as 6 – 6.5 months compared to 7-8 months for control. The useful dwarf, which considered as desirable traits, showed a balance of height and girth.
International Journal of Pharmaceutical and Biological Archive
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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).
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This review article deals with brief description of fat soluble vitamins with figures and tables
showing statistical analytical data duly quoting the references wherever necessary. The word “soluble” actually
means “able to be dissolved.” Whether a vitamin is classified as 'fat-soluble' or 'water-soluble' has to do with
how the vitamin is absorbed, stored and removed from the body. Vitamins are tiny organic compounds with a
huge impact on the health and well-being of the body. The body needs a small amount of fat soluble vitamins in
order to stay in optimal health. Fat soluble vitamins play an important role in keeping the body healthy and
functioning from immune system and muscle and heart function, easy flow and clotting of blood as well as eye
health. They are critical to health and wellness–particularly reproductive health and wellness. Low-fat, no-fat
and vegan diets are woefully lacking in fat soluble vitamins. However a diet based on traditional foods can
naturally provide these vitamins. Science is still learning about many of the functions of vitamins. "Too much
vitamin A, D, or K can lead to increased levels that are unhealthy and can cause serious health consequences.
Diseased conditions leading to decreased fat absorption leads to decreased absorption of vitamins. The fatsoluble
vitamins work most safely and effectively when obtained them from natural foods within the context of a
diet rich in all their synergistic partners. If fat soluble vitamins are stored for lengthy time they generate threat
for toxicity than water soluble vitamins and such situation even aggravated, provided they are consumed in
excess. Vitamin products, above the legal limits are not considered food supplements and must be registered as
prescription or non-prescription (over-the-counter drugs) due to their potential side effects. Vitamin A and E
supplements do not provide health benefits for healthy individuals, instead they may enhance mortality, and it is
held proved that beta-carotene supplements can be harmful to smokers
Sulphasalazine Induced Toxic Epidermal Necrolysis A Case Reportiosrphr_editor
Toxic Epidermal Necrolysis (TEN) is a rare and life threatening mucocutaneous reaction
characterized by extensive necrosis and detachment of epidermis. The Worldwide incidence of TEN is 0.9 to 1.4
per million populations per year [1]. Here we have discussed a case of Toxic Epidermal Necrolysis secondary
to Sulfasalazine managed with fluid replacement, analgesics, anti-infective therapy aggressive nutritional
support and intravenous high dose steroid therapy.
Keywords- Toxic Epidermal Necrolysis, Sulfasalazine
Evaluation the efficacy of IVIgG in treatment of Hemolytic Disease of Newborniosrphr_editor
Hemolytic disease of newborn (HDN) is an important cause of hyperbilirubinemia in the
neonatal period,and delayed diagnosis and treatment may lead to permanent brain damage. Traditional
neonatal treatment of HDN is intensive phototherapy and exchange transfusion.Intravenous
immunoglobulin(IVIgG) has been introduced as an alternative therapy to exchange transfusion. This study was
conducted to assess the effect of IVIG in HDN .
FIBROLIPOMATOUS HAMARTOMA OF ULNAR NERVE: A RARE CASE REPORT.iosrphr_editor
Nervous fibrolipomatous hamartoma is said to be a rare tumor-like condition involving the peripheral
nerves,in which the epineurium and perineurium are enlarged and distorted by excess of fatty and fibrous tissue
s that infiltrate between and around nerve boundaries. The median nerve is more likely to develop a hamartoma
than other nerves with a predilection for the carpal tunnel.
A fibrolipomatous hamartoma – is a rare, benign, congenital lesion most commonly found in the median nerve,
usually at the level of the wrist or hand.
We report a case of this rare condition in ulnar nerve.
SELF MEDICATION PRACTICES FOR ORAL HEALTH PROBLEMS AMONG DENTAL PATIENTS IN B...iosrphr_editor
Introduction: Self‑ medication is commonly practiced all over the world. Self-medication is defined as the use
of medication by a patient on his own initiative or on the advice of a pharmacist or a lay person instead of
consulting a medical practitioner. The present study was aimed to estimate the prevalence of self-medication for
oral health problems among dental patients in Bengaluru city; to identify triggering factors that could influence
self-medication practices; to identify sources of medications used; to identify sources of information about
medications used; and to identify reasons for self-medication.Study Design: A Cross sectional Study.Methods:A
survey was conducted among 175 subjects among dental patients in Bengaluru city. Data were collected
through a specially designed proforma using a closed‑ ended, self‑ administered questionnaire containing 15
questions, in five sections.
Results: The prevalence of
Clinico-haematological Profile of Falciparum Malaria in a Rural Hospital of T...iosrphr_editor
Aim: To study the clinico-haematological profile malaria in a rural hospital of Tripura.
Material and methods: A cross-sectional hospital-based study was done from at Kulai District
Hospital,Tripura. This hospital based cross sectional study was done on 60 confirmed cases of falciparum
malaria (either by peripheral smear or rapid diagnostic test) admitted in Kulai District Hospital. A case sheet
proforma was prepared and data (demographic profile,clinical feature, investigation, treatment, and
complication) from all indoor patients was collected and analyzed.
Result: Out of 60 patients, 40(66.6%) were males and 20 (33.4%) were females. Most of the patients were
between the age group 21-40 years with the highest prevalence between the age group of 21-30. Fever was the
most common symptom. Anemia was present in 42(70%) patients, out of which 6(10%) patients had severe
anemia. Thrombocytopenia was present in 36(60%) patients.Abnormal liver function tests were observed in
26(43.3%) subjects while abnormal kidney function tests were observed in16(26.6%) patients. All the 60
patients received Artemisinin based antimalarial drugs.
Conclusion: Early detection, prompt management, and adequate supportive therapy may reduce mortality due
to falciparum cerebral malaria.
Indonesian Wild Ginger (Zingiber sp) Extract: Antibacterial Activity against ...iosrphr_editor
Lempuyang gajah (Zingiber zerumbet (L.) Smith), lempuyang pahit (Zingiber amaricans BL.), and
lempuyang wangi (Zingiber aromaticum Vahl.) are used as traditional medicine (jamu) in Indonesia. It is also
used for treatment of microbial infections, helps to increase appetite and stimulate digestion in chickens.
Information on their uses are available, but only limited in the scientific data on their bioactivity. The study was
conducted on the antibacterial effect of organic extracts of these plants with Mycoplasma gallisepticum as the
agent of chronic respiratory disease in chickens. Juice and extracts of fresh and dried rhizome are evaluated
through the disc diffusion assay and minimum inhibitory concentration. Oxytetracyclin (30 µg) are used as
standards. All extracts are individually exhibited as antibacterial activity against Mycoplasma gallisepticum (7
± 0.11 mm to 21 ± 0.86 mm). The minimum inhibitory concentration (MIC) determination of plants extracts are
ranged from 7.8 mg/ml to 31.2 mg/ml. The preliminary results suggested promising antibacterial properties of
wild ginger from Indonesia, and probably could be used in management of chronic respiratory disease in
chickens.
A case of allergy and food sensitivity: the nasunin, natural color of eggplantiosrphr_editor
Abstract: Allergies and food sensitivities can both be considered as "adverse reactions individualistic" to food.
Are pathological and individual forms because they affect a few individuals in way rather serious; immediate
or delayed reactions occur instead with simple effects histamine, or, in severe cases with respiratory and
anaphylactic shock
The eggplant (Solanum melongena L.) is known to cause food allergies in some Asian countries, but detailed
studies on allergies caused by eggplant are lacking, however, it was highlighted the presence of allergens in
edible parts of eggplant with preponderance in the peel .
The purpose of this study was to propose an extraction method rapid, efficient and cost of natural dye from
waste products from the food industry, such as the peels of eggplant, from which it was extracted, isolated and
purified the nasunin,a colored molecule in red-fuchsia.
Nasusin was tested on 58 patients to evaluate the potential sensitizing effect on the skin. The results demonstrate
that allergenic effects are negligible and therefore the nasunin can be used as a colorant in various industrial
sectors with a certain safety margin
Complete NMR Assignment of MogrosidesII A2, II E andIII A1Isolated from Luo H...iosrphr_editor
NMR analysis allowed complete assignments of three known mogrol glycosides, Mogroside IIA2 (1),
II E (2)and IIIA1 (3), isolated from the extracts of Luo Han Guo. Herein, complete 1H and 13C NMR
assignmentsof all threemogrosidesare described based on NMR experiments (1H NMR, 13C NMR, COSY,
HSQC-DEPT, HMBC, NOESY and 1DTOCSY) and mass spectral data.
Nanoemulsion and Nanoemulgel as a Topical Formulationiosrphr_editor
: Nanoemulsion is referred type of emulsion with uniform and extremely small droplet size in the range
of 20-200 nm. Nanoemulsion provides numerous advantages over other carrier such as polymeric nanoparticle
and liposomes, including low cost preparation procedure, high hydrophilic and lipophilic drug loading system
to enhance the longer shelf live upon preserving the therapeutic agents. Incorporating the preparation of
nanoemulsion with hydrogel matrix to produce nanoemulgel exhibited by the two separate systems that forming
it. Nanoemulgel possesses the properties of thixotropic, non-greasy, effortlessly spreadable, easily be removed,
emollient, not staining, soluble in water, longer shelf life, bio-friendly, translucent and agreeable appearance.
Pharmacokinetics of High-Dose Methotrexate in Egyptian Children with Acute Ly...iosrphr_editor
Aim:Since several factors have been shown to influence the clearance of methotrexate, the purpose of this study
was to identify potential relationships between patient covariates and the methotrexate clearance estimates and
deduce a pharmacokinetic model for the estimation of methotrexate clearance in Egyptian pediatric ALL
patients that may help dosage adjustment and achieve target steady-state plasma concentrations in a similar
sittings.
Patients and methods: A total of 94 pediatric patients with B-cell ALL, of whom 70 were the studied population
and 24 were the test population, were treated with four courses of HDMTX doses 2.5 gm/m2
(low-risk arm) or 5
gm/m2
(standard-/high-risk arm) given every other week by intermittent intravenous infusions over 24 hours as
a part of their treatment protocol. Patients were monitored for the 24 hour MTX concentration and the systemic
methotrexate clearance was calculated for each methotrexate dose
Epidemiology of Tuberculosis (TB) in Albania 1998-2009iosrphr_editor
Abstract : In Albania, many people erroneously think that tuberculosis (TB) is a disease of the past-an illness
that no longer constitutes a public health threat. Surveillance is an integral part of tuberculosis (TB) control.
Albania has a highTB notification rate and there are doubts about underreporting. The evolution of the
incidence of tuberculosis is presented, together with more detailed figures over the period 1998-2009. These
figures were obtained by the monthly forms (called 14/Sh) compared with the individual notification data.
Objective: To examine the distribution and sources of increased tuberculosis (TB) morbidity and reporting
system deficiencies in the Albania from 1998 through 2009. Metodology: The study is descriptive one conductet
during the period 1998-2009. The statistical analysis is based on data reported from regional level (regional
epidemiological departments) to the central level (Public Health Institute). Results: The main findings were:
discordance between the collected data (individual form) and reported data (monthly form); tuberculosis
incidence rate shows little oscillations which ranges from 6.67 to 9.2 cases/100.000 population; 50% of the
regions show a lack of information on the confirmation of diagnosis and laboratory examination type used for
confirmation. Conclusion: TB disease in high-risk populations where it is difficult to detect, diagnose, and treat;
limitations of current control measures and the need for new tests and treatments, including an effective
vaccine; improving information system, regulation of individual form and personnel training.
Total Phenol and Antioxidant from Seed and Peel of Ripe and Unripe of Indones...iosrphr_editor
Study on total phenol and antioxidantactivity ofsugar apple fruits of various solvent, part of fruits, and level of ripening. Solvent extraction used were 80% (v/v) methanol, 50% (v/v) acetone, boiling water, and 50% (v/v) ethanol. Part of fruits thatbeen used for samples were seed and peel which are normally by products of sugar apple processing, level of ripening were unripe, and ripe sugar apple fruits. Total phenol was determined by Folin-ciocalteau method. Total antioxidant was quantified by 1,1-diphenyl-2-picrylhydrazyl(DPPH) method.Therewas a difference in type of solvent, part of fruits, and level of ripeningon total phenol and antioxidant concentration of sugar apple fruits. Seeds have higher total phenol concentration than peels of this fruits. Unripe sugar apple fruits have higher total phenol and antioxidant than ripe fruit. The best solvent for phenol extraction was ethanol 50%butthe best solvent for antioxidant extraction was acetone 50%.
A Review on Step-by-Step Analytical Method Validationiosrphr_editor
When analytical method is utilized to generate results about the characteristics of drug related samples it is essential that the results are trustworthy. They may be utilized as the basis for decisions relating to administering the drug to patients. Analytical method validation required during drug development and manufacturing and these analytical methods are fit for their intended purpose. To comply with the requirements of GMP pharmaceutical industries should have an overall validation policy which documents how validation will be performed. The purpose of this validation is to show that processes involved in the development and manufacture of drug, production and analytical testing can be performed in an effective and reproducible manner. This review article provides guidance on how to perform validation characteristics for the analytical method which are utilized in pharmaceutical analysis.
A Cross Sectional Study of Ethnic Differences in Occurrence and Severity of A...iosrphr_editor
Non-steroidal anti-inflammatory drugs are the most widely used "over the counter" medication all over the world despite their complications in different major organs. Present studies envisaged for knowing the occurrence and severity of adverse drug reactions from NSAIDs in different ethnic communities of Sikkim. A cross sectional study was undertaken in the medicine outpatients department of a secondary and tertiary care hospital. The patients belonging to Nepalese, Bhutias, Lepchas ethnic communities and others community (settlers from other parts of India) were included to analyzed the data based on the age and gender, ethnicity and ADRs, drugs and ADRs. Severity assessment was done using Hartwing and Siegel scale and causality assessment by Naranjo scale. Total 109 cases of ADRs, predominating in female were detected. Nepalese were the most affected and Gastrointestinal tract (GIT) being the most affected organ in them. Diclofenac showed maximum number of ADRs in all the communities. Maximum number of cases occurred on single day use (40.36%) of drugs. All the cases were belonging to the "possible category" and the maximum being the mild (72.48%) in nature. It is advisable to consider the ethnic/racial differences equally with other factors, to improve the safety and efficacy of a drug.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
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Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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A high frequency microcloning protocol for subsequent cryopreservation in Kaempferia galanga L.: An endangered, over exploited medicinal plant in pharmaceutics
1. IOSR Journal Of Pharmacy
(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219
www.iosrphr.org Volume 4, Issue 12 (December 2014), PP. 16-23
16
A high frequency microcloning protocol for subsequent
cryopreservation in Kaempferia galanga L.: An endangered, over
exploited medicinal plant in pharmaceutics
T.S. Preetha#*
, A.S. Hemanthakumar, P. Padmesh, P.N. Krishnan
Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute,
Palode, Thiruvananthapuram - 695562, Kerala, India
#
Department of Botany, University College, Thiruvananthapuram - 695034, Kerala, India
Abstract: An efficient in vitro propagation protocol was standardized in K. galanga, wherein shoot cultures
were raised from rhizome with axillary bud explants in Murashige and Skoog (MS) medium supplemented with
different hormonal regimes. Maximum 10.6±0.83 multiple shoots per explants were obtained in MS medium
supplemented with 4.0 mgl-1
6-benzyl adenine (BA) along with 1.0 mgl-1
each of α-naphthaleneacetic acid (NAA)
and kinetin. Subsequent 2-3 subculture passages enhanced the shoot multiplication rate to 3-4 times. The
multiple shoots thus produced were having individual root system and elongated substantially when kept in the
same medium for 4-6 weeks. There was no need of elongation and root induction phase and this two-step
multiplication procedure reduced the period of plantlet production compared to the earlier protocols. The
regenerated plants after a short hardening phase got established in the field at 80-90% efficiency and their
genetic uniformity was confirmed through ISSR analysis. The protocol thus standardized is an efficient method
for rapid propagation of this high value medicinal species by which at least 30 shoots per explants can be
produced within 12 weeks duration. It offers the possibility of raising physiologically uniform plants for easy
dissection of shoot tip meristems which can be efficiently utilized for subsequent long-term conservation through
cryopreservation.
Key words: endangered medicinal plant, in vitro clonal propagation, Kaempferia galanga
I. INTRODUCTION
Kaempferia galanga (Family Zingiberaceae) is an endangered medicinal plant [1] possessing several
therapeutic applications [2,3]. There is no seed formation naturally and conventional propagation is via
rhizomes which is dormant during drought and sprouting in spring. Always there is scarcity of propagules as
they constitute the commercial product of medicinal importance. Due to the demand for the drugs obtained from
rhizomes, the plants are over exploited, thereby threatening its survival. To meet the market demand and
considering the propagation problem, developing feasible protocols for rapid multiplication and effective
conservation are essential. In vitro preservation by cryopreservation ensures the long-term maintenance of plant
germplasm for which a reliable micropropagation system is necessary to obtain as many physiologically
uniform in vitro grown plantlets. Though in vitro propagation of K. galanga has been reported by several
authors [4,5,6,7,8,9,10,11,12] such established procedures does not reveal the production of a large number of
relatively homogenous shoot tips of uniform size, physiological state and growth response. The present study
provides a high frequency in vitro propagation system with the production of genetically uniform plants as
evident by ISSR banding pattern which can be subsequently utilized for cryopreservation of shoot tips thereby
increasing the chances of positive and uniform response to subsequent cryogenic treatments.
II. MATERIALS AND METHODS
2.1. Plant material
Kaempferia galanga plants (Herbarium voucher: TBG 60637) collected from the Kulathupuzha forest
range under Kerala Forest Department and maintained at the field gene bank of JNTBGRI served as the plant
material for the present study.
2.2. Culture establishment
Rhizomes with axillary buds collected from the field-grown plants were thoroughly washed under
running tap water, outer scales were removed using scalpel and washed in 3-4 drops of Teepol, dissolved in 100
ml distilled water for 20 minutes followed by washing in running tap water. After several rinses in distilled
water, the explants were subjected to sterilization with 0.1 % (w/v) HgCl2 for 8-10 minutes followed by 4-5
rinses in sterile distilled water. Axillary buds along with basal rhizome part (1 cm) excised aseptically were
2. A high frequency microcloning protocol for subsequent cryopreservation ……
17
implanted on MS medium [13] containing 0.6% (w/v) agar, 3% (w/v) sucrose and supplemented with 0.5 mgl-1
BA. The cultures were incubated for 4-6 weeks at 25±2 °C and 12 hour photoperiod with a light intensity of 50-
60 µEm-2
s-1
provided by cool, white fluorescent tubes (Philips India, Mumbai) under 50-60% RH.
2.3. Shoot multiplication, elongation and rooting
The initiated shoot buds were subcultured individually after 4 weeks to fresh medium augmented with
1.0, 2.0, 3.0 and 4.0 mgl-1
BA or kinetin either individually or 2.0, 3.0, 4.0 mgl-1
BA in combination with 1.0
mgl-1
kinetin or 0.5 and 1.0 mgl-1
NAA or IAA to enhance the multiplication, elongation and rooting. Scaling up
of shoot multiplication was done by repeated subculture of the shoot clumps after removing the outer scales
surrounding the individual shoots in selected hormonal combinations. Observations were recorded at regular
intervals. Data were statistically analyzed based on ANOVA and their significance was assessed using Duncan’s
Multiple Range test at p≤ 0.05 using SPSS software.
2.4. Hardening and field transfer
The plantlets weaned away from the culture vessels were washed thoroughly in running tap water to
remove traces of agar and treated with 0.1 % Dithane M-45 for 5 minutes to avoid fungal infection. They were
transplanted in polybags filled with potting medium of garden soil and river sand mixture (1:3) and hardened in
a mist house under constant irrigation at 28±2 ºC and 80±5% RH (M/s Indo-American Exports Ltd.) for 1-2
weeks. The hardened plants were then transferred to a shade net house under 50% sunlight and regular watering
for 3-4 weeks. Such established plantlets were transferred to the field during premonsoon and monsoon rains to
facilitate their establishment.
2.5. Genetic uniformity analysis using ISSR markers
Genomic DNA from leaf samples of regenerated plants were isolated using modified CTAB method
[14]. ISSR assay was carried out in 25 μL reaction mixture containing 0.2 mM dNTP’s, 10 mM Tris-HCL,1.5
mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 1.0 U Taq DNA polymerase (Finzymes, Helsinki, Finland), 15
pmol primers (IDT, Coralville, USA) and 50 ng of genomic DNA. The amplification was performed in a
thermal cycler (Eppendorf ESP-S). After the initial cycle of 2 min at 93°C, 2 min at 50 to 55°C (annealing
temperature of the primers ranges from 50 to 55°C for the different primers used in this study) and 2 min at
72°C. A total of 39 cycles of 1 min at 93°C, 1 min at 50 to 55°C and 1 min at 72°C were performed. The last
cycle was performed by 10 min extension at 72°C. Reaction mixture wherein template DNA replaced by
distilled water was used as negative control. Amplified products where resolved in 1.4% agarose gel (1xTBE)
followed by ethidium bromide staining. Amplification with each arbitrary primer was repeated 3 times and those
primers that produced reproducible and consistent bands were selected for data generation.
III. RESULTS
3.1. Culture establishment
Maximum of 76.67% infection free cultures were obtained from the rhizome explants which were
subjected to surface decontamination as described. Seasonal variation influenced the percentage of response.
Explants collected during rainy season (June-July) showed maximum contamination leading to a loss of up to
70% and those collected during summer season (January-April) showed minimum of contamination and loss
(30%) of explants.
Rhizomes with axillary buds of K. galanga inoculated on MS medium supplemented with 0.5 mgl-1
BA
induced direct shoot proliferation, while those transferred to medium devoid of growth regulators exhibited
elongation of existing primordium in limited number of explants. The explant responded after 15 days of culture
by breaking the outer thick sheath followed by emergence of shoot primordium (Fig.1a). The explants produced
1-3 shoots within 30 days. All the shoots developed were healthy with well developed leaves and one or two
roots.
3.2. Shoot multiplication
3.2.1. Effect of cytokinins on shoot multiplication
Single shoots subcultured on medium supplemented with different concentrations and combinations of
BA or kinetin either alone or in combination, or in combination with auxins exhibited differential response of
multiple shoot bud induction in 60-96.67% of the cultures. The multiplication rate was increased as the
concentration of cytokinins increased in the medium. After 30 days of subculture, MS medium supplemented
with 2.0, 3.0 and 4.0 mgl-1
BA produced an average of 4.89±0.74, 5.00±0.38 and 5.20±0.42 shoots respectively
(TABLE 1). Medium supplemented with 1.0-3.0 mgl-1
kinetin produced only 1-2 shoots within 30 days of
3. A high frequency microcloning protocol for subsequent cryopreservation ……
18
culture while an average of 2.71±0.28 shoots were obtained in 4.0 mgl-1
kinetin. No significant change in shoot
length was observed as the concentration of BA or kinetin increased.
3.2.2. Combined effect of cytokinins on shoot multiplication
Combination of BA and kinetin did not affect multiple shoot bud induction as that of BA alone, but
there was significant increase than with kinetin alone (TABLE 1). A linear increase in the production of
multiple shoots was noticed with increase in BA concentration in combination with kinetin. Combination of 1.0
mgl-1
kinetin and 2.0, 3.0 and 4.0 mgl-1
BA produced a mean number of 2.83±0.22, 3.14±0.26 and 5.75±0.38
shoots respectively (TABLE 1).
3.2.3. Effect of cytokinins in combination with auxins on shoot multiplication
BA in combination with auxins viz. NAA and IAA further increased the shoot multiplication rates and
the mean number of shoots varied from 3.8±0.29 to 7.4±0.65 (Fig. 1b) (TABLE 1). BA at a concentration of 2.0
and 3.0 mgl-1
in combination with 0.5 mgl-1
NAA/ IAA promoted shoot multiplication more or less equally. A
significant increase in the number of multiple shoot production was noted when 4.0 mgl-1
BA was used in
combination with 1.0 mgl-1
NAA (7.40±0.65 shoots) in comparison with the addition of 1.0 mgl-1
IAA
(6.00±0.35 shoots). All the shoots developed were healthy with well developed green leaves. A positive impact
on multiple shoot induction was noted when BA, kinetin and NAA were used together. MS medium
supplemented with 4.0 mgl-1
BA along with 1.0 mgl-1
each of kinetin and NAA induced the best shoot
multiplication (10.60±0.83 shoots) as well as the highest number of roots per shoot (Fig.1c) (TABLE 1). When
the concentration of each hormones was reduced to half (2.0 mgl-1
BA along with 0.5 mgl-1
each of kinetin/
NAA/ IAA) in the multihormonal combination tested, the rate of multiple shoot production was also reduced to
half.
3.3. Scaling up of shoot multiplication
In K. galanga, scaling up of shoot multiplication was achieved through repeated subculture of the shoot
clumps in those hormonal combinations which exerted the production of more than 5 shoots during the first
subculture period. Repeated subculturing for 1-2 cycles each of 4-5 weeks duration induced the production of an
increased number of multiple shoots. The shoot cultures responded with differentiation of buds from basal part
(resident meristem of the shoots) within 1-2 weeks. Relatively higher rate of multiplication was recorded in the
second passage compared to the first (TABLE 2). An average three-fold increase in the number of shoots was
observed during two subculture passages in almost all the growth hormone combinations tested. The shoot
clumps with maximum multiplication (10.60±0.83 shoots) obtained during first subculture in medium amended
with 4.0 mgl-1
BA and 1.0 mgl-1
each of kinetin and NAA produced 30.2±0.76 shoots after 60 days of culture
during the second subculture passage in the same media formulation (Fig. 1d) (TABLE 2). In this combination,
beyond two subculture passages (60 days), each subculture passage extended up to 4 weeks, a total of 30.2
shoots were harvested. Therefore, this hormonal combination was selected as the best for multiplication of shoot
buds in K. galanga. Thus it is possible to produce 30 shoots per explant starting from a single rhizome segment
within 60 days of inoculation.
During the third subculture, almost same rate of multiplication was observed after 90 days of culture
(TABLE 2). When the isolated shoots obtained in each subculture passage were frequently transferred to the
same media for a long period, shoot multiplication rates become reduced.
3.4. Shoot elongation and rooting
Plantlets developed in the multiplication medium elongated substantially within 40 days when left in
the same medium without subculture during the first subculture period itself and hence there was no need of
including the shoot elongation step. Moreover, root development was parallel with shoot multiplication in all the
combinations tested. Small roots were initiated from the basal part of the regenerated shoots after 15-20 days of
culture. The roots were green, thick and the number varied from 4-8. This is an advantage over only shoot
formation, where additional treatments are required for root induction.
The concentration of NAA and IAA affected the number of roots developing on subcultured plantlets
in K. galanga. The mean number of roots produced per shoot slightly increased as the concentration of NAA
and IAA increased in the medium. MS medium supplemented with 4.0 mgl-1
BA along with 0.5 mgl-1
NAA
produced 3.5 roots per shoot which was increased to 4.8 up on enhancement of the concentration of NAA to 1.0
mgl-1
. Increase in the rate of root induction was also observed in MS medium supplemented with 4.0 mgl-1
BA
along with 0.5 mgl-1
IAA, where 4.6 roots per shoot were produced; while supplementation of 4.0 mgl-1
BA
along with 1.0 mgl-1
IAA induced the formation of maximum 5.2 roots per shoot (TABLE 1). Root induction
was also observed even in MS medium supplemented with cytokinins alone.
4. A high frequency microcloning protocol for subsequent cryopreservation ……
19
3.5. Hardening and field transfer
The plantlets were successfully acclimatized in the mist house for a period of 1-2 weeks and then to a
shade net house for 3-4 weeks before transplanting into selected areas inside the JNTBGRI campus. Plantlets
with 7-8 cm height showed better establishment after hardening. The regenerated plantlets transplanted in
polybags showed 90-95% survival (Fig.1e) and got established in the field at 80-90% rate. They grew to mature
plants after 3-4 months of transfer, were free of any morphological variations and showed normal growth as
field grown plants in their yield, rhizome formation, etc. They were genetically uniform in ISSR analysis (Fig.
1f). Based on the results obtained, a schematic representation for in vitro clonal propagation of K. galanga is
presented in Fig.2.
IV. DISCUSSION
The rhizome explants were subjected to vigorous decontamination due to the underground, soiled
nature of the explants. Underground plant parts harbor different microorganisms and in such cases, multistep
disinfection process [15] is usually followed viz. initial wash with a detergent, followed by immersion in
commercial bleach, sodium or calcium hypochlorite and thereafter in 0.1% (w/v) HgCl2 for 5-10 minutes
depending on the nature of the tissue. The surface decontamination initially with teepol followed by aseptic
treatment with 0.1% HgCl2 for 8-10 minutes offered a reasonable decontamination protocol in K. galanga. For
successful decontamination, the explants had to be collected during summer months (January-March). The
infection-free axillary buds together with the basal rhizomatous part cultured on MS basal medium responded
with the elongation of the existing shoot tip with no additional proliferation of shoot buds. Exogenous supply of
cytokinins was essential to keep the apical bud alive and active.
In vitro-derived single shoots subcultured on medium supplemented with different concentrations and
combinations of growth regulators exhibited multiple shoot bud induction. BA played a significant role in
multiple shoot formation and gave better results compared to kinetin. MS medium supplemented with 4.0 mgl-1
BA produced an average of 5.20±0.42 shoots, where as 2.71±0.28 shoots were obtained in 4.0 mgl-1
kinetin. The
high performance of BA over other cytokinins for stimulation and proliferation of lateral bud growth was
reported earlier in K. galanga [7]. The effectiveness of BA on clonal multiplication of medicinal plants of
Zingiberaceae has been reported in Curcuma and Zingiber [16], Alpinia calcarata [17] and A. galanga [18].
However, an increase in the production of multiple shoots was noticed when kinetin was used in combination
with BA and a mean number of 2.83±0.22, 3.14±0.26 and 5.75±0.38 shoots were obtained in a combination of
1.0 mgl-1
kinetin along with 2.0, 3.0 and 4.0 mgl-1
BA. Thus combination of the two cytokinins increased
multiple shoot bud induction and this is in agreement with earlier findings [19]. Such a synergistic effect has
been reported by many workers in different plant species [20,21].
BA in combination with auxins further increased the shoot multiplication rates and the number of
shoots varied from 3.80±0.29 to 7.40±0.65 in MS medium supplemented with 2.0 mgl-1
BA in combination with
1.0 mgl-1
IAA and NAA respectively. Shirin et al. (2000) [7] showed that BA (2.7 mgl-1
) and NAA (0.5 mgl-1
)
was effective for the multiplication of K. galanga. The highest frequency of multiple shoot induction required a
specific ratio of auxin and cytokinin which varied with species and physiological status of the explants as
evident from earlier works on Curcuma zedaoria, K. galanga and K. rotunda [9,19,22,23]. Strong synergistic
effect of BA-NAA interactions on enhanced shoot production is also reported in banana also [24].
Scaling up of shoot multiplication was achieved through at least two subculture passages of 4 to 6
weeks each in media supplemented with 2.0 and 3.0 mgl-1
BA or 2.0-4.0 mgl-1
BA along with 1.0 mgl-1
NAA/
IAA. Maximum multiple shoots (10.60±0.83 shoots) obtained in MS medium supplemented with 4.0 mgl-1
BA
along with 1.0 mgl-1
each of kinetin and NAA after the first subculture period (30 days) increased to three times
(30.2±0.76 shoots) after 90 days of culture in fresh medium of the same combination indicating the additive
effect of this multihormonal combination in effecting multiple shoot induction. Once aseptic shoots are induced,
the successive multiplication rate gets maximized under in vitro conditions in most of the Zingibers [25]. The
plantlets were sufficiently elongated when left in the same medium during the first subculture period.
In K. galanga, irrespective of the growth regulator treatment healthy roots were induced with the
multiplication of shoots simultaneously. Hence there is no need of including separate root induction step, which
is an advantage in micropropagation of this species. Root induction in shoot inducing medium was reported in
other Zingibers. This may be due to intrinsic root inducing factors in the rhizome; and such a phenomenon has
been reported in rhizomatous plants [5]. Root induction was observed even in MS medium supplemented with
cytokinins alone which may be due to the increased endogenous auxin levels in the plant. There was no need of
media alternation for shoot elongation and root induction which enables easy propagation of the crop within a
short duration of time. Rooted plants hardened in the mist house for initial 4-6 weeks got established at
5. A high frequency microcloning protocol for subsequent cryopreservation ……
20
significant frequencies (90-95%) in pure sand and garden soil medium under irrigated conditions indicating the
feasibility of obtaining reasonable frequencies of establishment of K. galanga under less sophisticated
conditions.
The in vitro clonal plants established in the mist house when transferred to open field showed
conspicuous growth and emergence of new leaves. The introduction of the plants during the premonsoon
showers in late May or monsoon rains in early June facilitated easy establishment. The plantlets transferred to
selected areas inside JNTBGRI campus were free from morphological and growth abnormalities with high
establishment rate (80-90%) and showed uniform growth. This confirmed the utility of the methods for
conservation and revegetation purposes. The genetic stability assessed using ISSR markers established the
genetic fidelity of the regenerants.
Figure 1: Microcloning of K. galanga L. a) Shoot bud initiation in MS+0.5 mgl-1
BA; b) Multiple shoots in
MS+0.5 mgl-1
NAA+2.0 mgl-1
BA; c) Shoot multiplication in MS+4.0 mgl-1
BA+1.0 mgl-1
each of kinetin and
NAA; d) Multiple shoots in MS+4.0 mgl-1
BA+1.0 mgl-1
each of kinetin and NAA after 2 subculture passages;
e) Hardening of the plantlets inside the mist chamber; f) Monomorphic bands in ISSR analysis (M: Marker, 1-10
samples, C: mother plant)
Figure 2: Protocol for in vitro clonal propagation of K. galanga L.
Explants
(Rhizome with axillary buds)
MS+ 0.5 mgl-1
BA (4 wks)
Culture initiation
1-3 axillary shoots
10.6 plantlets
Subculture II
(60 days)
30.2 plantlets
Hardening (4-6 wks)
MS + 4 mgl-1
BA + 1 mgl-1
kinetin + 1 mgl-1
NAA
MS + 4 mgl-1
BA + 1 mgl-1
kinetin + 1 mgl-1
NAA
Surface sterilization
0.5% Teepol (20 min),
0.1%HgCl2 (8-10 min)
Subculture I
(30 days)
6. A high frequency microcloning protocol for subsequent cryopreservation ……
21
Mist/ Green house establishment (90-95%)
(3-4 months)
Field establishment (80-90%)
TABLE 1. Effect of plant growth regulators on shoot multiplication in K. galanga
Growth regulators (mgl-1
)
%
response
Mean no. of
shoots ± SE
Mean length of
shoots ± SE
Mean number
of roots ± SEBA KIN NAA IAA
1.0 - - - 73.33c
2.10±0.22g
3.19±0.15c
2.9±0.22e
2.0 - - - 76.67c
4.89±0.74e
3.57±0.26c
3.8±0.25d
3.0 - - - 70.00c
5.00±0.38d
3.07±0.19c
3.3±0.26d
4.0 - - - 80.00b
5.20±0.42d
3.41±0.24c
4.1±0.24c
- 1.0 - - 66.67d
1.40±0.15h
3.72±0.24c
2.0±0.21e
- 2.0 - - 63.33d
1.70±0.14h
3.10±0.16c
2.9±0.23e
- 3.0 - - 60.00d
1.91±0.21h
3.18±0.12c
2.8±0.20e
- 4.0 - - 63.33d
2.71±0.28g
3.68±0.13c
3.0±0.26d
2.0 1.0 - - 76.67c
2.83±0.22g
3.53±0.17c
3.3±0.21d
3.0 1.0 - - 73.33c
3.14±0.26f
3.54±0.12c
3.4±0.16d
4.0 1.0 - - 66.67d
5.75±0.38d
3.75±0.19c
3.5±0.17d
2.0 - 0.5 - 86.67b
4.10±0.18e
3.69±0.12c
4.6±0.22c
3.0 - 0.5 - 76.67c
5.14±0.31d
3.58±0.12c
4.7±0.39c
4.0 - 0.5 - 76.67c
6.14±0.26c
3.79±0.12c
4.6±0.26c
2.0 - 1.0 - 86.67b
5.57±0.41d
3.74±0.13c
4.8±0.29c
3.0 - 1.0 - 83.33b
5.62±0.47d
3.43±0.10c
4.9±0.31c
4.0 - 1.0 - 86.67b
7.40±0.65b
3.66±0.07c
5.2±0.29b
2.0 - - 0.5 86.67b
3.80±0.29f
3.64±0.10c
3.8±0.25d
3.0 - - 0.5 93.33a
4.14±0.39e
3.82±0.17c
3.3±0.26d
4.0 - - 0.5 90.00a
4.80±0.52e
3.83±0.07c
3.5±0.22d
2.0 - - 1.0 96.67a
4.87±0.29e
4.01±0.13b
4.6±0.30c
3.0 - - 1.0 83.33b
5.71±0.44d
4.17±0.20b
4.8±0.24c
4.0 - - 1.0 80.00b
6.00±0.35c
4.10±0.15b
4.8±0.29c
4.0 1.0 1.0 - 86.67b
10.60±0.83a
5.13±0.12a
5.6±0.37b
2.0 0.5 0.5 - 83.33b
5.40±0.84d
4.16±0.49b
4.1±0.85c
4.0 1.0 - 1.0 76.67c
7.90±0.54b
5.58±0.30a
5.0±0.30b
2.0 0.5 - 0.5 73.33c
4.40±0.31e
3.78±0.19c
6.1±0.41a
Data represents mean ± SE of 10 replicates repeated thrice; observations were recorded after 30 days of
culture. Means followed by the same letter in superscript in a column do not differ significantly based on
ANOVA and Duncan’s multiple range test (p ≤ 0.05).
TABLE 2. Effect of repeated subculture on shoot multiplication in K. galanga
Growth regulators (mgl-1
) Mean no. of shoots ± SE
BA KIN NAA IAA
Subculture I
(30 days)
Subculture II
(60 days)
Subculture III
(90 days)
Subculture IV
(120 days)
3.0 - - - 5.20 ± 0.42d
13.0 ± 0.62e
11.6 ± 0.52g
9.8 ± 0.39h
4.0 - - - 5.75 ± 0.38d
19.8 ± 0.92c
16.1 ± 0.48d
14.3 ± 0.71d
4.0 1.0 - - 5.00 ± 0.38d
12.0 ± 0.47f
10.3 ± 0.58h
9.3 ± 0.30h
2.0 - 0.5 - 5.57 ± 0.41d
11.7 ± 0.49f
10.1 ± 0.56h
8.9 ± 0.27i
3.0 - 0.5 - 5.62 ± 0.47d
15.1 ± 0.48d
13.1 ± 0.48e
10.6 ± 0.31g
4.0 - 0.5 - 6.14 ± 0.26c
18.3 ± 0.70c
15.9 ± 0.52d
14.5 ± 0.34d
7. A high frequency microcloning protocol for subsequent cryopreservation ……
22
2.0 - 1.0 - 4.87 ± 0.29e
13.0 ± 0.71e
11.5 ± 0.50g
11.0 ± 0.39f
3.0 - 1.0 - 5.71 ± 0.44d
17.5 ± 0.62c
16.2 ± 0.36d
14.9 ± 0.67d
4.0 - 1.0 - 7.40 ± 0.65b
20.7 ± 0.73b
18.4 ± 0.65c
17.1 ± 0.31c
2.0 - - 0.5 4.10 ± 0.18e
12.8 ± 0.66e
12.0 ± 0.65f
10.1 ± 0.46g
3.0 - - 0.5 5.14 ± 0.31d
16.3 ± 0.58d
13.9 ± 0.52e
12.4 ± 0.37e
4.0 - - 0.5 6.00 ± 0.35c
18.0 ± 0.61c
16.4 ± 0.54d
14.5 ± 0.40d
4.0 1.0 1.0 - 10.60 ± 0.83a
30.2 ± 0.76a
27.5 ± 0.52a
24.8 ± 0.39a
4.0 1.0 - 1.0 7.90 ± 0.54b
22.7 ± 0.80b
21.5 ± 0.73b
18.1 ± 0.40b
Data represents mean ± SE of 10 replicates repeated thrice; observations were recorded after 30 days of
culture. Means followed by the same letter in the superscript in a column do not differ significantly based on
ANOVA and Duncan’s multiple range test (p ≤ 0.05).
V. CONCLUSION
K. galanga is a clonally propagated medicinal plant having no seed set and a shoot tip cryopreservation
protocol is essential for its long-term conservation. Shoot tips are widely used for cryopreservation, because
they are totipotent, contain organized tissue and are genetically stable. For successful cryopreservation of plant
species, a reliable micropropagation system is necessary to obtain many in vitro grown plantlets. The in vitro
propagation system established here ensures the production of a large number of relatively homogenous shoot
tips in terms of size, physiological state and growth response as it is possible to raise at least 30 shoots per
rhizome bud, thereby increasing the feasibility of the procedure to subsequent cryotreatments which has also
been successfully accomplished [26]. The in vitro preservation and micropropagation thus ensures the
conservation and sustainable utilization of this endangered pharmaceutically important plant species.
Acknowledgements
We are thankful to the Director, JNTBGRI for providing the facilities. We also acknowledge the
financial assistance from Department of Biotechnology, Government of India as a part of ‘National Gene Bank
for Medicinal and Aromatic Plants project’ and Council of Scientific and Industrial Research, Government of
India for the award of CSIR-SRF to TS Preetha.
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