Large-scale Multiplication Research Articles

Effect of liquid medium on shoots amplification, in vitro flowering and ex vitro rooting of Oldenlandia umbellata L. - A dye yielding medicinal herb

Oldenlandia umbellata L. gains importance due to its medicinal properties and the presence of anthraquinones based natural dyes in the roots. Present study describes the effect of Murashige and Skoog’s (MS) liquid medium (full strength) on in vitro regeneration, flower bud induction and ex vitro rooting in O. umbellata. Shoot segments with 2-3 nodes (each node with 2 axillary buds) served as explants for establishment of cultures. The liquid medium augmented with 2.0 mg L-1 6-benzylaminopurine (BAP) with additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) was effective for shoot bud induction (6.4±0.19 shoots per explant within 2-3 weeks). The shoots were further multiplied (89.3±1.07 shoots, 2-3 weeks) when the shoot clusters obtained from the culture initiation directly transferred to the full-strength MS liquid medium incorporated with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 acetic acid (IAA) with additives. Flower buds were induced (12.0±0.15 buds per shoot) when the shoots were cultured on 1.0 mg L-1 BAP and kinetin (Kin, 6-furfurylaminopurine) and 0.5 mg L-1 of IAA at 45 µmol m−2 s−1 SFPD (Spectral Flux Photon Density) light intensity for 14/10h (light/dark) photoperiod. The adventitious roots were induced on 1/4 strength MS medium supplemented with 1.5 mg L-1 indole-3 butyric acid (IBA). Ex vitro rooting was achieved (16.0±0.53 roots per shoot) by pulse treatment of the shoots with 300 mg L-1 IBA for 2 min. The in vitro produced plantlets were acclimatized in the greenhouse and finally translocated to the in vivo conditions with 93 % success rate. This is the foremost (use of liquid MS medium) and cost-effective method for large scale multiplication of O. umbellata.

Effect of liquid medium on shoots amplification, in vitro flowering and ex vitro rooting of Oldenlandia umbellata L. - A dye yielding medicinal herb

Oldenlandia umbellata L. gains importance due to its medicinal properties and the presence of anthraquinones based natural dyes in the roots. Present study describes the effect of Murashige and Skoog’s (MS) liquid medium (full strength) on in vitro regeneration, flower bud induction and ex vitro rooting in O. umbellata. Shoot segments with 2-3 nodes (each node with 2 axillary buds) served as explants for establishment of cultures. The liquid medium augmented with 2.0 mg L-1 6-benzylaminopurine (BAP) with additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) was effective for shoot bud induction (6.4±0.19 shoots per explant within 2-3 weeks). The shoots were further multiplied (89.3±1.07 shoots, 2-3 weeks) when the shoot clusters obtained from the culture initiation directly transferred to the full-strength MS liquid medium incorporated with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 acetic acid (IAA) with additives. Flower buds were induced (12.0±0.15 buds per shoot) when the shoots were cultured on 1.0 mg L-1 BAP and kinetin (Kin, 6-furfurylaminopurine) and 0.5 mg L-1 of IAA at 45 µmol m−2 s−1 SFPD (Spectral Flux Photon Density) light intensity for 14/10h (light/dark) photoperiod. The adventitious roots were induced on 1/4 strength MS medium supplemented with 1.5 mg L-1 indole-3 butyric acid (IBA). Ex vitro rooting was achieved (16.0±0.53 roots per shoot) by pulse treatment of the shoots with 300 mg L-1 IBA for 2 min. The in vitro produced plantlets were acclimatized in the greenhouse and finally translocated to the in vivo conditions with 93 % success rate. This is the foremost (use of liquid MS medium) and cost-effective method for large scale multiplication of O. umbellata.

Phytochemical composition and antioxidant properties of prickly pear (Opuntia ficus-indica L.) flowers from the Algerian germplasm

Prickly pear Opuntia ficus-indica L. is a widely used plant in traditional medicines and as edible food product mainly in arid regions. Its phytochemical properties are primarily due to secondary metabolites present in its different parts. Quantity and quality of different metabolites and their associated biological activities are influenced by the genetic structure of medicinal plants and environmental conditions to which plants are exposed. This study aimed to assess the aqueous extract of O. ficus-indica flowers belonging to six populations from the Algerian germplasm in terms of phenolics, flavonoids, tannins, sugars and minerals contents along with the antioxidant activity revealed by the free radical scavenging activity (DPPH) and ferric reducing power assays. Results revealed that populations from high altitudes ‘Ain Defla’, ‘Msila’ and ‘Tiaret’ were characterized by their low phenolics content along with high flavonoids and sugars contents. However, populations from low altitudes showed high contents of polyphenols and condensed tannins but less sugars. In addition, all populations demonstrated a variable antioxidant activity. Variation in antioxidant properties and bioactive compounds contents among O. ficus-indica populations must be used for harnessing the maximum potential of the species and for breeding programmes at large-scale multiplication for commercial cultivation. The obtained results demonstrated the importance of Opuntia flowers as a potential source of bioactive substances for food and medicines.

Micropropagation, plantlets production estimation and ISSR marker-based genetic fidelity analysis of Guadua magna and G. angustifólia

ABSTRACT The bamboo species Guadua magna and G. angustifolia have been propagated nearly exclusively by conventional techniques of vegetative propagation. Micropropagation is a promising technique and an alternative to conventional ones. This study aimed to micropropagate plants, estimate the plantlets production and analyze the genetic fidelity of G. magna and G. angustifolia by ISSR molecular markers. Mother plants of both species were cultivated in a greenhouse, and either sprayed or not with fungicide. In the laboratory, microcuttings were disinfested and established in MS culture medium with 3.0 mL L-1 of Plant Preservative Mixture (PPM®) and 1 mL L-1 of Carbendazin®. The contamination-free shoots were multiplied in liquid or semi-solid MS medium with 3.0 mg L-1 of BAP for five subcultures. Rooting was performed in liquid or semi-solid MS1/2 medium, plus 3.0 mg L-1 of IBA. Acclimatization was performed on a commercial substrate, in a growth chamber, and the genetic fidelity of the clones produced was analyzed via ISSR markers. The addition of fungicide and PPM® to the medium reduced the contamination in G. magna, but not in G. angustifolia. The liquid medium was more efficient than the semi-solid one for the multiplication of both species, which showed production potentials between 760 and 920 plants per initial microcutting, after five subcultures. Rooted plants exhibited a survival rate of up to 100 % in acclimatization. No polymorphic regions were found in the clones analyzed by ISSR at the end of the fifth subculture, suggesting that micropropagation is a safe technique for the large-scale multiplication of bamboos.

Micropropagation of Gynostemma pentaphyllum by internode culture techniques

Gynostemma pentaphyllum is a rarely valuable medicinal plant that people has been traditionally used for disease treatment in a long time. The modern medical studies have also shown that it exhibits a variety of pharmacological properties including anti-inflammatory, antioxidative, lipid metabolism regulatory, antiproliferative, neuroprotective and anxiolytic activities. However, conservation and exploiting this medicinal plant were not managed properly and studies of biotechnology on this medicinal plant were till limited. Therefore, the application of plant cell biotechnology in conservation and development of G. pentaphyllum is necessary. The internode segments of G. pentaphyllum were sterilized with diluted solution of javel (50%) for 20 minutes. The rate of sterile explants reached to 73.33%. In vitro shoots tips and cutting stem segments of G. pentaphyllum were used as explants and cultured on MS medium supplemented with BA (0.1; 0.5; 1.0; 1.5; 2.0 mg/L) combined with NAA (0; 0.1; 0.2; 0.3 mg/L) for shoot proliferation. After 6 weeks, new shoots were generated and the MS medium containing 1.0 mg/L BA and 0.1 mg/L NAA gave the highest shoot induction (6.8 shoots/explant). To determine the mineral media suitable for growth of G. pentaphyllum, regenerated shoots were cultured on different mineral media. The MS ½ medium was suitable for growth of shoots with 5.2 cm height and 4.0 leaves/plantlet. For root induction, the MS ½ medium supplemented with 0.25 mg/L IBA was optimal, the root length could be in 7.6 cm in this medium. This system could be utilized for large-scale multiplication of G. pentaphyllum.

In vitro mass propagation and conservation of a rare medicinal plant, Zhumeria Majdae Rech.f & Wendelbo (Lamiaceae)

Abstract The monotypic Zhumeria majdae (Lamiaceae), an endangered medicinal and aromatic plant in the south part of Iran, has a low propagation rate in natural condition and therefore an efficient method for the in vitro-propagation is required. In the present study, the effects of source and position of the explants as well as various concentrations of 6-benzylaminopurine and kinetin on inhibition of hyperhydricity and in vitro mass propagation of the plant were investigated. The best shoot formation (1.9 ± 0.07) was obtained with a Murashige and Skoog medium fortified with 17.7 µM 6-benzylaminopurine. Regenerated shoots, elongated on the MS medium containing 2.2 μM 6-benzylaminopurine, were rooted on the different tested media, with the most abundant (68.6 ± 4.1%) and strongest roots obtained on half-strength medium without plant growth regulators. Hydro-distilled essential oils of in vitro regenerated plant were analyzed by gas chromatography–mass spectrometry and compared with the essential oil of wild plant. Thirty and twenty compounds representing 98.2% and 98.7% of the total oils were identified in the oils of wild plant and in vitro regenerated plant, respectively. The major essential oil components were linalool (31.7% and 41.7%), camphor (28.0% and 32.4%), limonene (4.6% and 8.3%), camphene (4.1% and 3.5%) and E-caryophyllene (1.0% and 2.7%) in the studied (wild plant and in vitro regenerated plant) essential oils. The in vitro regeneration system could be utilized for both conservation, large-scale multiplication and production of rich linalool essential oils of Z. majdae.

PLANT REGENERATION FROM CALLUS CULTURES DERIVED FROM MATURE ZYGOTIC EMBRYOS OF SOPHORA ALOPECUROIDES LINN. IN MONGOLIA

The aim of this study is to determine the effect of hormones and selection of the most effective medium using callus cultures derived from mature zygotic embryos of Sophora alopecuroides Linn. for plant regeneration. After 8 weeks of culture, the highest callus induction medium (93.3%) was obtained on MS medium supplemented with 0.2 mglL Zeatin and 2.0 mg/L α-naphthaleneacetic acid (NAA). The best callus proliferation was observed on the same medium. Shoots regenerated at the highest frequency of 50.0% with 5.8 shoots when calli were cultured on MS medium with 2.0 mg/L BA. Therefore, this protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of S.alopecuroides L.

Seed germination and vegetative production of Greyia radlkoferi, an indigenous tree of cosmeceutical importance

Greyia radlkoferi is one of the important and recently studied cosmeceutical trees or shrubs indigenous to South Africa. It offers great potential as a source of extracts for the development of cosmeceutical products with the ability to treat hyperpigmentation. In order to develop the product, G. radlkoferi must be produced on a commercial scale. Greyia radlkoferi is known to regenerate naturally by seed. In an effort to introduce G. radlkoferi into cultivation, the effect of five temperature treatments ranging from 10-30°C on the germination of its seeds was investigated. The trial was carried out under laboratory-controlled conditions in incubators in continuous darkness. Germination of G. radlkoferi seeds was discovered to be dependent on temperature. Warm temperatures between 20 and 25°C were more favourable for the germination of this species with optimum germination temperature being 25°C with 82% germination. In vegetative propagation by stem cuttings, the effect of cutting position, exogenous rooting hormone (Seradix® 1, Seradix® 2, 0.1, 0.3 and 0.8% IBA) and the cutting position were studied in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings rooting better with 35% rooting compared to 6% rooting attained for the apical cuttings. Exogenous hormones did not show any statistical significant effects on the rooting of cuttings, however, a clear trend in response to the growth regulator treatments was observed, with 0.1% IBA recording the highest rooting percentage. Considering the results of this study, seed propagation appears to be the suitable technique for large-scale multiplication of G. radlkoferi. This data contribute to the establishment of an efficient protocol for cultivation of G. radlkoferi for commercial purposes.

Indirect Organogenesis through Seedling-Derived Leaf Segments of Ficus Religiosa - a Multipurpose Woody Medicinal Plant

The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.

In vitro biotechnological advancements in Malabar nut (Adhatoda vasica Nees): Achievements, status and prospects.

Adhatoda vasica Nees, belonging to family Acanthaceae, is a well-known medicinal plant. It is endorsed for its pyrroloquinazoline alkaloids and its derivatives, such as vasicine and vasicinone. Germinating A. vasica seeds is a tedious task; on that account, vegetative propagation is the preferred method for its multiplication. For rapid and large-scale multiplication, germplasm conservation as well as secondary metabolites production, in vitro culture of A. vasica was preferred over conventional propagation by several researchers; however, some major applications of this tissue culture technique are still awaiting to undergo extensive research. The present review, for the first time, illustrates all the major achievements associated with in vitro regeneration of A. vasica, reported till date and highlights the future prospects.

Improving Macropropagation and Seed Germination Techniques for Conservation of Threatened Species

Populations of threatened plants are declining rapidly in natural habitats due to various anthropogenic activities. Reinforcement of the dwindling populations through reintroduction is a promising aspect for conservation of threatened plants. However, due to lack of standardized propagation methods of such plants, mass production of planting materials has become a challenge, thereby constraining the replenishment process. Identification of factors constraining the seed germination of threatened plants and addressing it effectively, are among the most cost-effective strategies for large-scale multiplication and subsequent conservation of the threatened species. Similarly, conventional low-cost vegetative propagation techniques such as grafting, air layering, and regenerating plantlets from root-suckers, apical meristems, and stem cuttings often prove more successful for multiplication than relatively costly micropropagation techniques. In this article, we present a few case studies on low-cost mass propagation techniques of threatened plant species of India through seed, stem/apical shoot cutting and airlayering, that helped in the restoration of the species.

Embryogenesis followed by enhanced micro-multiplication and eco-restoration of Calamus thwaitesii Becc.: an economic non-wood forest produce for strengthening agroforestry system

The present study is focussed on development of a high-frequency micro-multiplication system in Calamus thwaitesii, through somatic embryogenesis from immature zygotic embryos cultured in Murashige and Skoog (MS) medium supplemented with 31.68 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The semi-friable calli when cultured in the same medium augmented with 2.22 µM 6-benzylaminopurine (BAP) and 1.07 µM α-naphthalene acetic acid (NAA) induced ~ 12 discrete globular embryoids in 6 weeks. The isolated embryoids in hormone-free media yielded 65% plantlets. Furthermore, embryoids and axenic shoots exhibited maximum shoot induction in medium supplemented with 0.45 µM Thidiazuron (TDZ). The shoot initials after subculture in media supplemented with 1.78 µM BAP and 0.45 µM TDZ produced shoot proliferation followed by elongation in basal medium. The elongated shoots produced roots in media supplemented with 16.11 µM NAA. With this established protocol, ~ 5940 rooted plantlets could be harvested after 40 weeks from a single embryoid. Genetic stability analysis of the plantlets using inter simple sequence repeat markers recorded only 0.05% genetic polymorphism. The plantlets were hardened in a mist house for 8 weeks, and then to 50% shade house for another 16 weeks, and the well-established 6-month-old nursery plants, reintroduced to selected forest segments, exhibited 86% field establishment even after 3 years of observation. Thus, the mass multiplication system developed could be a breakthrough for large-scale multiplication of C. thwaitesii to ensure continuous supply of quality planting material to the cottage industry through the development of agroforestry systems. Furthermore, the in vitro culture system developed here can be replicated for research activities related to the long-term–short-term conservation, micro-multiplication and sustainable utilization of rare, endangered, endemic, monopodial/single stemmed rattan palms.

Direct and indirect shoot organogenesis strategies for propagation and conservation of Abutilon ranadei Wooder & Stapf - A critically endangered plant.

The study aimed to evaluate the efficiency of direct and indirect organogenesis for large-scale multiplication, propagation and conservation of Abutilon ranadei, an endemic and critically endangered plant. Nodes and leaves from in vitro germinated seedlings were used as source of explants for direct and indirect shoot organogenesis respectively. High frequency shoot organogenesis (77.5%) with 7.1 ± 0.2 shoots were achieved when nodes were cultured on MS medium with BAP (3.0 mg/l) and GA (0.5 mg/l). The best callus induction and proliferation response was achieved with 2,4-D (2.0 mg/l) and BAP (1.5 mg/l). Maximum response (92.5%) for shoot regeneration from callus with 13 ± 0.8 number of shoots were obtained in MS medium fortified with KN (1.5 mg/l) and NAA (0.4 mg/l). Microshoots regenerated via direct as well as indirect organogenesis were rooted best in ½MS containing 1.5 mg/l IBA and 1.0 mg/l BAP. The well rooted plantlets were successfully established in the soil.

EFFECT OF COCONUT WATER AND ACTIVATED CHARCOAL ON SEED GERMINATION IN AN ENDEMIC ORCHID RHYNCHOSTYLIS RETUSA BLUME.

The present study on in vitro seed germination of Rhynchostylis retusa obtained on nutrient medium made clear pathway for the in vitro propagation of orchids by seed culture. The presence of coconut water in the medium enhanced seed germination and growth of seedlings. For enhancing the growth of seedlings further the individual seedlings were cultured on MS medium supplemented with 3 mg/l BAP and 1g/l activated charcoal. These seedlings were sub cultured on rooting medium (IBA) supplemented with activated charcoal. The plantlets were transferred to soil with 90% success. Hence the present investigation made possible for the large scale multiplication of R.retusa and thus to conserve this endemic orchid.

Micropropagation of an Important Medicinal Plant, Begonia fallax (Begoniaceae)

Begonia fallax A.DC. belongs to the family of Begoniaceae and is an endemic species to Peninsular India. Shoot tip and nodal explants were screened using Murashige and Skoog medium supplemented with BAP, KIN and TDZ individually and combination with IAA, NAA and IBA for multiple shoot initiation. BAP and NAA (0.5+0.5 mg-l) produced 20.8 shoots with up to 4.0 cm shoot length to nodal explants whereas 17.9 shoots with 3.1 cm shoot length to shoot tip explants. For root induction, various concentrations of IAA, NAA and IBA were used individually. The highest rooting frequency was achieved to nodal explants (64.90 roots) on MS medium supplemented with 0.5 mg-l NAA compared to shoot tip explants (31.90 roots). Invariably, root length was up to 2.3 cm to both the explants. The rooted explants were transplanted into plastic cups and showed 70% survival rate in the Environmental Growth Chamber. Nodal explants could be the best for large-scale multiplication and propagation to facilitate in vitro conservation and for secondary metabolites production.

Arbuscular mycorrhizal fungi enhance antioxidant capacity of in vitro propagated garden thyme (Thymus vulgaris L.)

Garden thyme (Thymus vulgaris L., Lamiaceae) is an important aromatic herb used for its medicinal values including antioxidant and antimicrobial properties. The present study was performed to analyze the changes in natural antioxidants after inoculation of in vitro propagated garden thyme plants with arbuscular mycorrhizal fungi (AMF). An efficient and low-cost protocol for large-scale multiplication of this aromatic plant was developed. The explants were cultured on full and half strength Murashige and Skoog (MS) medium containing indole-3-butyric acid (IBA). The maximum number of shoots and roots was obtained on ½ MS medium supplemented with 0.1 mg L−1 IBA after 4 weeks of culture. The successfully adapted in vitro plants (survival rate 95%) were inoculated with arbuscular mycorrhizal fungi (Claroideoglomus claroideum, ref. EEZ 54). Plants were then transferred into field conditions. Mycorrhizal fungi enhanced the activity of some soil enzymes, acid and alkaline phosphatase, urease as well as the levels of extractable glomalin-related proteins in plant rhizosphere. Arbuscular mycorrhizal associations with higher plants promote the accumulation of antioxidant metabolites such as phenols and flavonoids and increase the activity of antioxidant enzymes. The results from the present study suggest enhanced antioxidant capacity of the inoculated T. vulgaris plants which was due mainly to increased accumulation of phenolic compounds (total phenols and flavonoids) together with stimulation of the activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPO).

Evaluation of the growth and sporulation of different entomopathogenic fungi in different liquid and solid media at varied concentrations

Entomopathogenic fungi like Beauveria bassiana, Metarhizium anisopliae and Lecanicillium lecanii are used in biological control of agricultural insect pests. Their specific mode of action makes them an effective alternative to the chemical Insecticides. Virulent strains of Entomopathogenic fungi are effectively formulated and used as bio-insecticides world-wide. Amenable and economical multiplication of a virulent strain in a large scale is important for them to be useful in the field. Culture media plays a major role in the large-scale multiplication of virulent strains of Entomopathogens. Different substrates and media components are being used for this purpose. Yet, each strain differs in its nutritional requirements for the maximum growth and hence it is necessary to standardize the right components and their optimum concentrations in the culture media for a given strain of Entomopathogen. In the current study, three different nitrogen sources and two different carbon sources were tried to standardize the mass multiplication media for seven test isolates of Entomopathogenic fungi. A study was also conducted to determine the ideal grain media for the optimum conidial yields of the test isolates. Yeast extract was found to be the best Nitrogen source for the isolates. The isolates tested, differed in their nutritional requirements and showed variation in the best nitrogen and carbon sources necessary for their growth. Variation was also found in the optimum concentration of both the ingredients for the growth and sporulation of the isolates. In the solid-state fermentation study, rice was found to be the best grain for the growth of most of the fungi followed by barley. The significance of such a study in the development of an effective Myco-insecticide is vital and can be successfully employed in agriculture is discussed.

The channels of groundwater and river water as a torsion field waveguides

A set of factors and processes that have any kind of negative effect or impact on living organisms resulting from scientific and technological progress of mankind are said to be techno-pathogenic effect. The problem of the survival of humanity and the biotech protect all life on our planet is becoming one of the most important. The anthropogenic pollution of water, air and soil plays an essential role in the degradation of our environment, everyone agrees. We can also see the multiplication of installations linked to electricity: Mobile, telephone, wind, Wi-Fi, Bluetooth, high voltage lines; all these technologies have improved our daily life, but they can have a negative impact, invisible. The article that we are about to read may surprise us because it looks at problems whose origin is not well known and which are rarely taken into account. Yet these problems are real and it is logical to think that the recent and large-scale multiplication of antennas and wind turbines with their grounding induces fields that alter the natural equilibrium of the soil and have effects on the biosphere. It should be noted that awareness of the reality of these phenomena is progressing, but more for human health than for animals. It was determined, that the installation of masts base stations and wind turbines at the intersection of the fault zones, which is a natural migration of groundwater or geopathogenic zone, causes the appearance of artificial radiation of these zones and the spread of radiation occurs along areas faults or discontinuities along the axial in the soil. Constant exposure to natural and artificial radiations can cause changes in behavior, the state of aggression or depression (sometimes to the point of suicide), constant fatigue, sleep problems, partial loss of memory, problems with concentration, stress, etc. All electronic devices generate like natural cosmogenic fields-technical torsion field. Large amount of free radicals are produced in the body under their influence. To prevent a negative impact on the living it is necessary to harmonize the physical vacuum. The action of these harmonization devices is based on the elimination of the asymmetry of physical vacuum components. The simple harmonizing device consists of several elements, which were recorded the left and the right torsion field, before we have developed the device of the physical vacuum harmonization. When the device is a plug in the powerline network the physical vacuum is harmonized. It is necessary to pay much attention to theoretical issues of influence of technical torsion field on the human body and alive and biomedical research, one of the main areas of studying natural and technical torsion fields must be brought to a new level. Scientists, medicines and governments must move immediately and aggressively with the goal of minimizing the impact of torsion fields on children, pregnant women and adults.

DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Micropropagation of Caralluma adscendens var. fimbriata-An Indigenous Medicinal Plant of India

Present study describes micro-propagation of traditionally important medicinal plant in northern interior part of Karnataka, Caralluma adscendens var. fimbriata, for its conservation. Results showed that the survival ability of regenerated plants was 84 per cent. Proliferation of C. adscendens var. fimbriata shoots was achieved on medium supplemented with various concentrations of different hormones. The effective combination of 6-Benzylaminopurine (BA) 2.0 mg/l along with 0.5 mg/l kinetin (Kn) and 0.2 mg/l Indole-3-acetic acid (IBA) produced highest number of shoot sprouting frequency (91%) with a mean of 2.60 ± 0.17 shoot number per explants and a mean shoot length of 3.88 ± 0.20 cm from the mature nodal explants. A good frequency of callus was induced in 1.5 to 2.0 mg/l of 1-Naphthaleneacetic acid (NAA) and in 2.0 mg/l of 2,4-dichlorophenoxy acetic acid (2, 4-D) were transferred to root induction media. The NAA showed positive response of rooting when compared with IAA and IBA in the present study. The best root and shoot sprouted plants were successfully acclimatized in the greenhouse and transferred to open field conditions. This micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant.