Half Murashige And Skoog Medium Research Articles

In vitro propagation and DNA barcoding of the rare near endemic Plantago sinaica (Barnéoud) plant in Saint Katherine, Sinai

Plantago sinaica is a rare perennial shrub near-endemic to Egypt and found in Saint Katherine Protectorate in Sinai. The first successful in vitro propagation protocol was conducted to protect the plant outside its natural reserves. Shoot tip, stem node section, cotyledonary node, and root explants separated from in vitro germinated seedlings were cultured in vitro on Murashige and Skoog (MS) medium enriched with different concentrations and types of cytokinins. It was found that 6-benzyl adenine (BA) is the most efficient cytokinin. MS medium containing 3.33 µM BA and 0.54 µM α-naphthalene acetic acids (NAA) produced 10.25 and 11.30 shoots/explant using shoot tip and stem node section, respectively. Conversely, MS medium + 2.22 µM BA + 0.54 µM NAA produced 13.25 shoots from root explants. Surprisingly, the cotyledonary node explants favored MS medium free from plant growth regulators (PGRs), which produced only 4.25 shoots/explant. The multiplied shoots were rooted successfully with a 100% rooting percentage on half MS medium containing 1.23 or 2.46 µM indole-3-butyric acid (IBA). In vitro, rooted plantlets were efficiently transferred to the greenhouse with a 90% survivability. Finally, the plant was identified using three DNA barcodes; 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), plastid photosystem II protein D1 intergenic spacer region (psbA–trnH), and Internal Transcribed Spacer (ITS) barcodes. Additionally, psbA–trnH and ITS were novel and submitted to the GenBank databases for the first time for Plantago sinaica. Our study supports the United Nations Sustainable Development Goal number 15, which is to preserve, restore and reinstate sustainable usage of terrestrial ecosystems and to stop biodiversity loss.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.

In Vitro Regeneration of Korarima (Aframomum corrorima (Braun) P. C. M. Jansen): A Threatened Spice and Medicinal Herb from Ethiopia.

Developing an in vitro regeneration system is very important to increase production and productivity of plants as well as for the conservation of rare and threatened medicinal plants like korarima (Aframomum corrorima (Braun) P. C. M. Jansen). To date, no study dealing with in vitro indirect regeneration system of korarima has been reported. Thus, in this study, we developed an efficient and reproducible protocol for in vitro regeneration of korarima via callus. The procedure involved soaking seeds in 50% H2SO4 for 16 h that resulted in 92.5% germination on plant growth regulators (PGRs)-free half-strength Murashige and Skoog (MS) basal medium after a month. Shoot and rhizome induction rate of 93.75% was obtained on the MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA after five weeks. Whitish yellow friable callus was obtained from rhizome culture taken from in vitro grown plantlets. The MS medium containing 2.0 mg/l 2, 4D in combination with 0.5 mg/l kinetin, resulted in 77.5% callus induction. The shoot regeneration rate of 45% was obtained from callus on the MS medium containing 2.0 mg/l TDZ in combination with 0.5 mg/l IBA. The mean shoot number of 10.83 per explant was obtained upon multiplication on the MS medium containing 1.5 mg/l BAP with a mean shoot height of 5.37 cm. The best rooting responses were obtained on half MS medium supplemented with 0.5 mg/l IAA resulting in a mean number of root of 18.59, mean root length of 9.71 cm, and mean shoot height of 7.32 cm. The plantlets showed 75% survival efficiency after acclimatization. The present regeneration protocol offers a conceivable system towards effective conservation and genetic improvement of the crop by increasing the efficiency of genetic transformation.

In vitro propagation of an economically important medicinal plant Lawsonia inermis L. through nodal segments

The present investigation aimed to standardize efficient plant regeneration protocol through in vitro culture by using nodal segment for mass multiplication of Lawsonia inermis an economically important medicinal plant species. Mass multiplication of shoots induced on Murashige and Skoog (MS) medium supplemented with different growth regulators like auxins and cytokinins separately and in different combinations. The medium fortified with 6-Benzylaminopurine ( BAP) 1.0 mg/l + kinetin (KN) 1.5mg/l explained best compared to all other combinations. In vitro raised plantlets were excised and transferred in half strength MS medium supplemented with different growth regulators like Indole Butyric acid ( IBA) and naphthalene acetic acid (NAA ) (0.5-3.0 mg/l) in an experiment that gave rise to rooting. The half strength of MS medium additive with IBA in separate and in different combinations with NAA concentrations (0.5-3.0 mg/l) supported root development. The best response of rooting was obtained on half MS medium fortified with 1.0 mg/l IBA. The regenerated plantlets were successfully transplanted to pots. Regenerants were transferred to the field conditions and recorded the survival rate.. Among all the carbon sources and gelling agents used, sucrose (3%) in combination with 0.8 per cent agar-agar has proved significantly better. Multiple shoots formation with longer shoots were achieved on medium with 1.0mg/l BAP and 1.5mg/l Kn. Thus, it is possible to develop a large number of plants of L. inermis through shoot bud regeneration which can cater for the need of pharmaceutical as well as other industries.

Modified basal culture medium improves proliferation of Dendrobium Sabin Blue’s protocorm-like bodies (PLBs)

This study was carried out to reformulate half-strength Murashige and Skoog (MS) basal medium, one of the standard media for orchid tissue culture for protocorm-like bodies (PLB) culture of Dendrobium Sabin Blue. Initial PLB fresh mass of 0.5 g was inoculated in various culture treatments for six weeks of culture period. Alteration of half MS medium yielded a modified medium without Fe-EDTA and containing half-strength macronutrients, double-strength micronutrients, full-strength vitamins and 30 g/L sucrose. The modified medium enhanced the proliferation of PLBs by 23% (0.299 ± 0.017 g) as compared half MS medium (0.243 ± 0.012 g) on a dry mass basis. PLB suspension culture was established using liquid medium of the revised formula for a period of 7 weeks. Supplementing the modified medium with quarter-strength of Fe-EDTA is beneficial for long-term culture of PLBs. Improvement of PLB biomass through the use of modified culture medium devoid of plant growth regulators reduces production cost for micropropagation and secondary metabolite production. Besides, it can reduce the likelihood of somaclonal variations related to the use of plant growth regulators in the culture medium.

Hormonal Regulation for <i>de novo</i> Organogenesis in <i>in vtro</i> Derived Leaf and Internode of <i>dAegle marmelos</i> L.

Adventitious shoot formation from internode explants of Aegle marmelos was achieved. Leaf and internode explants were procured from shoots obtained from in vitro germinating seedlings. Callogenesis was induced from leaf and internode explants on Murashige and Skoog (MS) semisolid medium variously supplemented with 6-Benzyladenine (BA) and 2, 4-Dicholophenoxyacetic acid (2, 4-D) while the organogenic response was found remarkable only in BA supplemented medium. Both types of explant markedly influenced callus formation while organogenesis was noticed only in internode explants. The number of shoots formed in internode explants was greater in the medium supplemented with 0.5µM BA and registered 6.86 numbers of shoot. The regenerated shoots transferred to half MS medium supplemented with 2mg/l IBA produced 75% rooting followed by 66% in IAA supplemented medium. The plantlets raised in vitro were successfully hardened to ex vitro conditions.

Micropropagation of Arid Zone Fruit Tree, Pomegranate, cvs. ‘Malase Yazdi’ and ‘Shirine Shahvar’

ABSTRACT An optimum culture medium for micropropagation of pomegranate, cvs. ‘Malase Yazdi’ and ‘Shirine Shahvar’ is defined and protocols have been developed for in vitro shoot proliferation and rooting of the cultivars. Axillary buds of young shoots were used as explants. The shoots were multiplied in vitro on Murashige and Skoog (MS) supplemented with 0, 1.5, 2, and 2.5 mg.l−1 of N6-benzyladenine (BA). The micropropagated/cloned shoots were rooted on MS and half strength (1/2) of MS containing indole-3-butyric acid (IBA) at three concentrations (0, 0.5, and 1 mg.l−1) and 0.5 mg.l−1 naphthalene acetic acid (NAA). The cultivar ‘Malase Yazdi’ multiplied better than ‘Shirine Shahvar’. In both cultivars, the maximum shoot number with maximum shoot height was produced on medium with 2 mg.l−1 BA. Control medium free from plant growth regulator also supported shoot multiplication. The cultured shoots exhibited the greatest number of leaves on a medium containing 2.5 mg.l−1 BA. The cloned shoots of ‘Malase Yazdi’ rooted the best on half-strength MS medium with 0.5 mg.l−1 IBA while shoots of ‘Shirine Shahvar’, rooted on 1 mg.l−1 IBA and 0.5 mg.l−1 NAA in half MS medium. This method can be used to quickly propagate and conservation of these two pomegranate cultivars for future prospects.

Hairy root culture optimization and resveratrol production from Vitis vinifera subsp. sylvesteris

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.

NAA-Induced Direct Organogenesis from Female Immature Inflorescence Explants of Date Palm.

Micropropagation has great potential for the multiplication of female and male date palms of commercially grown cultivars by using inflorescences. This approach is simple, convenient, and much faster than the conventional method of using shoot-tip explants. We describe here a stepwise micropropagation procedure using inflorescence explants of Iraqi date palm cultivar Maktoom. Cultured explants were derived from 0.5-cm-long spike segments excised from 8 to 10-cm-long spathes. About 70% formed adventitious buds on Murashige and Skoog (MS) medium supplemented with 2mg/L naphthalene acetic acid (NAA), 4mg/L benzylaminopurine (BAP), and 40g/L sucrose and maintained in the dark for 16weeks before transferring to normal light conditions. The best multiplication rate was achieved with 3mg/L 2ip and 2mg/L; for shoot elongation, the best medium is MS containing 0.5mg/L BAP, 0.5mg/L 2ip, and 1mg/L GA3. Well-developed shoots were cultured for rooting in half MS medium amended with 1mg/L NAA and 45g/L sucrose. Plantlets with well-developed roots were successfully hardened in the greenhouse. Inflorescence explants proved to be a promising alternative explant source for micropropagation of date palm cultivars.

Direct Organogenesis from Rhizome Explants in Marsilea quadrifolia L.: A Threatened Fern Species

An efficient micropropagation protocol has been developed for Marsilea quadrifolia L. through direct organogenesis. The mature rhizomes were used as explants and successfully sterilized using 0.1% HgCl2 for the establishment of cultures. The multiple shoots were differentiated from the explants on Murashige and Skoog (MS) medium augmented with 6-benzylaminopurin (BAP). Full strength MS medium was reported to be effective for the induction of sporophytes from the rhizomes after four weeks of inoculation. Maximum response (96%) with average of 6.2 shoots (2.72 cm length) was achieved on full strength of MS medium augmented with 0.5 mg/L BAP in culture initiation experiments. The cultures were further proliferated in clusters (79.0±0.37 shoots per explant) with stunted growth on half strength MS medium supplemented with 0.25 mg/L BAP after four weeks. These stunted shoots were elongated (5.30 cm long) on half MS medium devoid of growth hormones. Root induction and proliferation (3.0–4.0 cm long) were observed after 4th subculture of sporophytes on hormone-free half strength MS medium. The rooted plantlets were hardened in the fern house for 4-5 weeks and transferred to the field with 92% survival rate. There were no observable differences in between in vivo grown and in vitro propagated plantlets in the field.

Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets

Cattleya intermediais an Atlantic Forest species endemic to Brazil that is classed as vulnerable on the list of threatened species. In this study, C. intermedia plantlets were micropropagated in an asymbiotic culture and the influence of different concentrations of sucrose (15, 30, 45 and 60 g L-1, plus a zero sucrose medium) and macronutrient salts (complete Murashige and Skoog (MS) medium and half MS medium (with half-strength macronutrients)) on survival and development of the plantlets was evaluated. In all treatments 100% plantlet survival was achieved. The integrated analysis of height of aerial part, number of leaves per plantlet, fresh mass, number of roots per plantlet and length of the longest root showed that the plantlets exhibited greatest development at the half-strength macronutrient concentrations with 45 or 60 g L-1 of sucrose, as well as at the complete macronutrient concentration with 60 g L-1 of sucrose. Plantlets acclimatized and reintroduced to an environment in which the species occurs naturally exhibited 98.6% survival. The results obtained in this study allowed the establishment of optimal conditions for asymbiotic micropropagation, which is a requisite for future studies focused on conservation of C. intermedia.

MICROPROPAGATION OF SOME NEW PEACH ROOTSTOCKS

Two experiments were conducted in 2011 and 2012 seasons on two newly introduced peach rootstocks, namely Garnem and Cadaman (which are widely used in Europe) in addition to the wellknown Nemaguard peach rootstock for comparison. The aim of this study is to optimize tissue culture protocol for propagation of the rootstocks under study. Results indicated that Nodal cuttings were better than shoot-tips in regard to aseptic culture (without contamination after 10 days) during establishment stage. However after one month, the three rootstocks gave rise to 100% survival percentage. Meanwhile number of leaves on Nemaguard rootstock was significantly the greatest. During proliferation stage, Garnem produced the highest shoot number, while Cadaman rootstock produced significantly the lowest shoot number. Regarding the effect of BA supplementation, it was found that BA at 2.0 ml/l resulted in the longest shoots. During rooting stage, 2.0 mg/l GA3 significantly gave the longest shoots. At root formation, there were no significant differences between the 3 rootstocks concerning root number , rooting percentage and root length. 0.1 mg/l IBA incorporated in half MS medium strength gave the highest significant values for the same traits. During acclimatization stage, Garnem rootstock achieved the highest significant survival ratio, while half strength Murashige and skoog (MS) medium resulted in better survival ratio, plant height and number of leaves. Sand + peat moss potting mixture achieved better survival ratio, plant height and number of leaves than Sand + Vermiculite. Protein banding patterns profile data proved the presence of band with molecular weight 20.094 k Da in Garnem rootstock, which may be responsible for high number of shoots and survival ratio. This band was absent in other rootstocks.

English

The present study was initiated to optimize in vitro protocol for mass propagation of two commercial sugarcane clones (Co 449 and Co 678) grown in Ethiopia through shoot tip culture. Experiments on shoot multiplication and rooting were laid out in a completely randomized design with factorial treatment arrangements. Shoot tips were surface sterilized with 5% active chlorinated Berekina for 25 min and initiated on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 6- benzylamino purine + 0.5 mg L-1 indole-3- butyric acid. For in vitro multiplication, aseptically initiated shoot tips were treated with different concentrations and combinations of BAP and Kin using either sucrose or table sugar in separate experiments. For root induction, regenerated shoots were transferred onto half MS medium supplied with 6% sucrose or table sugar and different concentrations and combinations of IBA and NAA. With regard to shoot multiplication, genotype Co449 showed maximum regeneration frequency of 80% with 7.87 ± 1.06 shoots per explants on MS medium with 3% sucrose and 2 mg L-1 BAP + 0.25 mg L-1 Kin. On the same carbon source, genotype Co678 showed the highest multiplication frequency of 90% with 9.10 ± 0.10 shoots per explant on medium supplied with 2 mg L-1 BAP + 0.5 mg L-1 Kin. On MS with 4% table sugar and 2 mg L-1 BAP + 0.25 mg L-1 Kin, 80% of the transferred explants of genotype Co449 produced multiple shoots with an average number of 7.61 ± 0.10 shoots per explant while genotype Co678 showed the highest regeneration frequency (86.67%) with mean shoots number per explant of 8.36 ± 0.04 on medium supplemented with 2 mg L-1 BAP + 0.5 mg L-1 Kin. Half MS + 6% sucrose + 2.5 mg L-1 IBA induced the highest rooting (86.67%) with an average root number per shoot of 16.53 ± 0.02 in Co449 cultures. In genotype Co678, ½ MS + 6% sucrose + 5 mg L-1 NAA induced the highest rooting response of 80% with an average root number per shoot of 13.17 ± 0.29. Equivalent rooting responses were recorded on half MS medium supplemented with 6% table sugar. On½ MS with 6 % table sugar, 2.5 mg L-1 IBA induced the highest rooting response (86.67%) and root number (15.93 ± 0.81) in genotype Co449 while 5 mg L-1 NAA gave the maximum (80%) rooting with average roots per shoot of 13.93 ± 0.81 in genotype Co678. Rooted shoots were transplanted in the green house for hardening and different survival rate was recorded.   Key words: Saccharum officinarum, multiplication, rooting, sucrose, table sugar, in vitro. &nbsp

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96% of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0mg l(-1)) in combination with 6-benzylaminopurine (BA; 0.8mg l(-1)). For callus regeneration, various concentrations of BA (1.0-5.0mg l(-1)) or thidiazuron (TDZ; 1.0-5.0mg l(-1)) alone or in combination with indole-3-acetic acid (IAA; 0.2-1.0mg l(-1)) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0mg l(-1)) and IAA (0.5mg l(-1)) combination. Here, 100% cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.

A Simple and Efficient In Vitro Mass Multiplication Procedure for Stevia rebaudiana Bertoni and Analysis of Genetic Fidelity of In Vitro Raised Plants Through RAPD

An investigation was carried out to develop an efficient micropropagation protocol for Stevia rebaudiana Bertoni. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Out of the different media compared for in vitro shoot multiplication, hormone-free liquid medium was found the most suitable. The results also revealed that strength of Murashige and Skoog (MS) salt, culture medium consistency (solid or liquid) and type of culture vessel significantly influenced the in vitro shoot multiplication rate. The shoots when placed on half strength hormone free half MS medium and 100 ppm charcoal showed cent percent root induction with maximum number of roots per shoot (21.2) as well as maximum root length (4.22 cm). Hardening of rooted plants on biopeat showed more than 80 % of survival rate. Further, clonal fidelity of the in vitro raised plants was carried out using RAPD markers and results indicated that all the tissue cultured derived plants are true-to-type and there are no somaclonal variations among these plants.

Shoot organogenesis from leaf callus and ISSR assessment for their identification of clonal fidelity in Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant

An efficient plant regeneration protocol via shoot regeneration from callus derived from immature leaf explants was established for Rhinacanthus nasutus (L.) Kurz., a potent ethnomedicinal plant for various ailments including cancer. 5-day old leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA; 1.0–5.0mg/l) or Kinetin (Kn; 1.0–5.0mg/l) in combination with 0.5mg/l IBA for callus induction. Optimum morphogenic callus induction (98.8%) was observed on MS medium supplemented with 4.0mg/l Kn and 0.5mg/l IBA. The excised callus subcultured on MS medium supplemented with BA (1.0–5.0mg/l) or Kn (1.0–5.0mg/l) with NAA (0.2 and 0.5mg/l). The highest frequency of shoot regeneration (96.3%) and shoot number (36.5 shoots per 1.0 gm calli) was observed on MS medium supplemented with Kn (3.0mg/l) in combination with NAA (0.5mg/l). Half MS medium supplemented with IAA (2.0mg/l) produced an optimum rooting response of 96.23% with an average number of 6.1 roots per shoots. Out of 50 rooted shoots transferred to soil 47 survived after acclimatization. Clonal fidelity of the regenerated plants with mother plant was evaluated using 12 inter simple sequence repeats (ISSR) primers and genetic uniformity among all the regenerants and mother plant is confirmed.

Callus regeneration from stem explants of Pseudarthira viscida (L.) Wight and Arn. - a vulnerable medicinal plant.

An in vitro propagation protocol has been developed for Pseudarthira viscida (L.) Wight and Arn. (Papilionaideae), a vulnerable medicinal plant from stem callus was used for axillary shoot multiplication. The plant was standardized by using MS (Murashige and Skoog) medium containing 3% (w/v) sucrose, supplemented with a cytokinin (BAP, KN) maximum number of shoot (29.87±5.34) were observed on the medium containing 0.6 mg/l BAP after three weeks of culture. Single shoots were induced and elongated on MS medium supplemented with BAP+NAA (0.4 + 0.04 mg/l). The elongated shoots were rooted in half MS medium with 0.4 mg/l IBA, and then were successfully hardened and transferred to the field.

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots.