Half Strength MS Liquid Medium Research Articles

Establishing a simple selection method of high hypericin-producing adventitious root lines from leaf explants of Hypericum perforatum var. Topaz

ABSTRACT Hundreds of adventitious root lines from Hypericum perforatum L. were examined for their hypericin content in order to establish a system for studying the production of hypericin in adventitious root lines. The effects of the medium consistency (solid and liquid) and the effects of three different auxins (Indole-3-butyric acid (IBA), Indole-3-acetic acid (IAA), and 1-Naphthaleneacetic acid (NAA)) on the induction of adventitious roots in leaf explants were studied. In a half-strength MS liquid medium with 2 mg/L IBA concentration, adventitious roots developed from H. perforatum leaves, whereas NAA and IAA caused callus-like and low branch roots. HPLC-UV was used to profile hypericin content variations in 100 H. perforatum adventitious root lines. Hierarchical component analysis (HCA) was used for selecting elite lines. After cluster analysis and selecting high-performance lines in terms of hypericin, five elite lines were chosen and the amount of hypericin production, as well as the phenol and flavonoid content, was analysed.

Clonal propagation of Dracaena fragrans cv. Victoria through tissue culture technology

Micropropagation of Dracaena fragrans cv.Victoria was conducted using the young, tender and disease-free leaves and nodal segments as explants collected from the local market of Savar, Dhaka. Surface sterilization of the explants pretreated with a liquid detergent and then 0.2% HgCl2 for 4-5 minutes produces maximum contamination free explants without any toxicity. After surface sterilization, different explants were inoculated on gelrite gelled MS medium supplemented with different concentrations of 2,4-D for callus induction and with different concentrations and combinations of BAP and NAA for direct shoot induction. Nodal explants showed high callus induction potentiality (80%) on MS medium supplemented with 1.5 mg/l 2,4-D. The highest frequency of direct shoot induction from nodal segment was 80% and the number of shoots per nodal segment was(5.28±1.17) when they were cultured on MS medium supplemented with 3.0 mg/l BAP and 0.3 mg/l NAA. The highest shoot multiplication (83.33%) with maximum number of shoot per unit callus (5.62±1.24) and maximum shoot length (3.27±0.82 cm) was observed when the nodal calli were transferred in gelrite gelled MS medium in combination with 4.5 mg/l BAP and 0.5 mg/l NAA. Additionally, the incorporation of 4% sucrose and 10% coconut water with the above mentioned medium showed the satisfactory shoot growth and development with an average 7.84±1.30 shoots per unit of callus which was 4.21±0.78 cm in length. Moreover, addition of 3.0 mg/l GA3 with the above mention medium showed highest rate of shoot elongation (5.83±2.31cm). For root induction, in vitro raised shoots were transferred onto half-strength of MS liquid medium augmented with different concentrations and combinations of auxins (IBA and NAA). Maximum rooting (75%) were observed in halfstrength MS liquid medium supplemented with 0.5 mg/l IBA. After appropriate rooting the plantlets were successfully acclimatized (85% survival) when they were cultured in polybag containing (1:1:1) garden soil, sand and compost mixture before transferred to soil. Regenerated plants were morphologically identical with mother plants and showed their uniform growth in field condition.
 Jahangirnagar University J. Biol. Sci. 8(2): 1-11, 2019 (December)

Impact of Antioxidants on in vitro Rooting and Acclimatization of Two Egyptian Dry Date Palm Cultivars.

Date palm (Phoenix dactylifera L.) is propagated vegetatively via offshoots, which is limited by either the offshoots numbers produced from a superior selected plant, or the occurrence of these offshoots only during the juvenile phase of the date palm life cycle. As a result, the in vitro propagation could be considered an alternative technique for large scale propagation of date palm. Obtaining well-developed root system is considered the most important step in establishment of reliable protocol for successful production of date palm and subsequently successful acclimatization of the in vitro derived plants. The aim of this study is to obtain a well-developed root system through using different antioxidants, with detecting the similarity between the in vitro derived plants and the mother plants using RAPD, long RAPD and ISSR techniques. Individual plantlets obtained from maturation of somatic embryos in vitro of about 5-7 cm in length with 2-3 leaves were used as plant material. Plantlets were cultured on half strength MS liquid medium supplemented with 0.5 mg L-1 thiamine-HCl+2.0 mg L-1 glycine+0.1 mg L-1 biotin+40 g L-1 sucrose+ 0.1mg L-1 NAA with different concentrations from either AgNO3 or citric or ascorbic acids (0.0, 0.5, 1, 2 mg L-1). Growth development, root number and root length (cm) were evaluated at the end of the second subculture (12 weeks). Data were reported as Mean±Standard deviation (SD). Data were subjected to one way-analysis of variance (p<0.05). Results were processed by Excel (2010). Among the different antioxidants with different concentrations used, generally it was found that 2 mg L-1 of each agent gave the highest values of growth development, roots number and roots length. However, using 2 mg L-1 AgNO3 gave the best results with all parameters. Regarding the response of date palm cultivar, it was remarkable that Bartamoda showed relatively better results than Sakkoty cultivar. According to PCR reactions, the results of RAPD, long RAPD and ISSR profile of tissue culture-derived plantlets grown on a medium supplemented with 2 mg L-1 AgNO3 obviously revealed high similarity to mother plants. It could be concluded that using 2 mg L-1 AgNO3 gave the best results for growth development, root numbers and length of the two cultivars but Bartamoda showed relatively better results than Sakkoty cultivar. The tissue culture-derived plantlets on this medium (2 mg L-1 AgNO3) revealed high similarity to mother plant as a result to RAPD, long RAPD and ISSR profiles.

In vitro propagation of Boerhaavia diffusa L.: An important medicinal plant of family Nyctagimaceae

Boerhaavia diffusa L., also known as santhi, or punarnava is an important medicinal plant, belonging to the family Nyctaginaceae. This species is said to be distributed throughout Malwa plateau in central India, as per ayurvedic literature, but due to extensive commercial exploitation, the species has become vulnerable. For callus induction, leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4- dichlorophenoxy acetic acid (2, 4-D 2.26 µM-9.04 µM) and N 6-benzyladenine (BA 1.11 µM-4.44 µM), either alone or in combinations. Calli formed within 10-12 days of culture, followed by shoots regeneration within 20-25 days. Direct organogenesis was achieved from nodal explants in MS media fortified with 2,4-D (2.26 µM-9.04 µM) along with BA (1.11 µM-4.44 µM) within 20 days. Multiple shooting was observed during subculture of in vitro regenerated shoots when 2,4-D was replaced with α-naphthalene acetic acid (NAA). Rooting was achieved in MS medium fortified with 2.85 µM IAA, within 7-10 days and also on half strength MS medium containing 2.85 µM Indole-3-acetic acid (IAA). For hardening, regenerated plants, with roots (3-4cm) were initially maintained on half-strength MS liquid medium (MS1/2) without growth regulators followed by quarter strength MS (MS1/4) liquid medium for 10 days. For acclimatization sterile mixture of soil, sand and manure (2:1:1) was used. Survival rate of regenerated plants was nearly 100%.

English

The study was aimed to standardise a protocol for the in vitro mass propagation of Rhinacanthus nasutus (L) Kurz, an anticancer shrub. Leaf, node and inflorescence explants were inoculated onto the Murashige and Skoog’s (MS) medium enriched with different combinations and concentrations of growth regulators. Maximum callusing percentage was achieved in leaf explants in MS medium supplemented with 5 mg/l 2,4-D. Multiple shoots were achieved from leaf, node and inflorescence explants with maximum of 25±0.42 (5 mg/l BAP + 2.5 mg/l IAA), 11±0.87 (5 BAP + 2.5 mg/l IAA) and 8±0.56 (3 mg/l BAP + 1.5 mg/l IAA) shoots, respectively. For in vitro rhizogenesis, elongated micro shoots were aseptically transferred to the half strength MS liquid medium with maximum number of 8±0.89 roots per shoot achieved in 1 mg/ml IAA fortified MS medium. The in vitro rooted micro shoots were acclimatised under laboratory conditions for two weeks by transferring to polycups containing sterile soil, sand and vermiculite (1:1:1). After two weeks, hardened plantlets were transferred to the green house for two weeks and then finally to the garden with 95% survivability. Key words: Rhinacanthus nasutus, multiple shoots, leaf callus, node, inflorescence, rhizogenesis.

Biosynthesis of Camptothecin from Callus and Cell Suspension Cultures of Ophiorrhiza mungos L. var. angustifolia (Thw.) Hook. f.

Ophiorrhiza mungos var. angustifolia is a herbaceous plant distributed in the southern western ghats of India. The species is extensively uprooted for the extraction of high-value anticancer alkaloid camptothecin (CPT), resulting in the eradication of natural population. The present work aims to develop an in vitro protocol for the sustainable production of CPT from callus and cell suspension cultures of the species to conserve the natural resource. Leaf explant excised from in vitro shoot cultures was inoculated onto solid half-strength Murashige and Skoog medium, fortified with auxins like Indole acetic acid (5.71–26.83 µM), Indole butyric acid (4.92–24.6 µM), Naphthalene acetic acid (5.71–28.54 µM), 2,4 Dichlorophenoxyacetic acid (4.52–22.62 µM) and Picloram (4.14–20.70 µM); either individually or in combination with Benzyl adenine, to raise the callus. Of the different combinations of auxins, 10.74 µM Naphthalene acetic acid + 4.44 µM Benzyladenine was selected as the best combination to produce maximum fresh weight of callus. The CPT content of callus was estimated as 0.23 ± 0.03 mg/g DW. Friable calli produced were used to raise cell suspension cultures in half strength MS liquid medium, fortified with different concentrations of auxins. Maximum fresh weight was yielded by a medium supplemented with 16.12 µM Naphthalene acetic acid (14.64 ± 2.03 g). Highest CPT content was estimated in a medium supplemented with 10.74 µM Naphthalene acetic acid as 0.31 ± 0.01 mg/g DW. When compared with the CPT content of field plants, it was found to be higher.

Elicitor mediated adventitious root culture for the large-scale production of anthraquinones from Oldenlandia umbellata L.

Anthraquinones (AQs) have an increasing global demand due to its importance in both industrial and pharmaceutical fields. Increased demand can be met with large-scale production of the compound. Enhanced production of AQs was achieved through elicitor mediated adventitious root cultures of Oldenlandia umbellata L. Adventitious root cultures were raised by incorporating different auxins into the half strength MS liquid medium. The root cultures were incubated for a period of 60 days with continuous monitoring at 10 days interval. Among the different auxins used, 1 μM indole 3 acetic acid (IAA) produced highest amount of total AQ (11.09 mg g−1 dry weight). Later, for enhanced production of AQs, elicitors viz., yeast extract, pectin, xylan, or precursors like α-keto glutaric acid and L- phenylalanine and an AQ inhibitor, piroxicam were added at a concentration of 10, 25 and 50 mg L−1 to the medium on 35th day of incubation and roots were harvested at 40th day of incubation. Addition of 50 mg L−1 pectin resulted highest (24.33 mg g−1 dry weight) elicitation of AQs from adventitious roots. HPLC analysis of samples from adventitious roots confirmed the presence of alizarin in the extract. By adopting adventitious root culture through pectin (50 mg L−1) as elicitor, 2.19 fold enhanced AQ production over control was achieved in O. umbellata.

Notes on In-vitro Propagation of Tecomella undulata (Sm.) Seem

Different explants of Tecomella undulata (Sm.) Seem. cultured on modified MS media gave significant differences for shoot regeneration and root differentiation. Plantlets were successfully developed using axillary bud and cotyledon as explants. There was no shoot formation from leaf explants. Maximum multiple shoots from axillary buds and cotyledons were obtained on medium containing MS major salts with BAP 4.0 mg/l + IAA 0.1 mg/l and BAP 5.0 mg/l + NAA 0.5 mg/l, respectively. Healthy roots developed from two step procedure. Isolated shoots were rooted by culturing on half strength MS liquid medium having IBA 2.5 mg/l for 48 hrs and then transferring to hormone free half strength solid MS medium. An initial dark period for 5-7 days favoured root induction. About 55 percent of in-vitro produced shoots were rooted.

Cost Effective Techniques for In Vitro Propagation of Two Varieties of Sugarcane

The present study was carried out to optimize cost effective in vitro protocol for mass propagation of two commercial sugarcane varieties grown in Maharashtra, India. The effect of different cytokinins and auxins on in vitro initiation, multiplication and rooting was investigated in sugarcane (Saccharum spp.) varieties; Co86032 and CoC671. Shoot tip explants comprising of apical meristems and 1-2 leaf primordia were carefully excised from the apical region and inoculated on MS basal liquid medium supplemented with different concentrations and combinations of Kinetin and BAP. Only initiation medium was sterilized by autoclaving and further shoot multiplication, rooting was done on medium sterilized with sodium hypochlorite (4% w/v available chlorine) at the concentrations 0.05% corresponding to total active chlorine concentration 0.002% in the medium. With regard to shoot initiation, variety Co86032 showed maximum shoot formation (96.67%) on MS medium supplemented with BAP (0.2mg/L) and Kinetin (0.1mg/L) with 3% sucrose and 10% CW on the other hand variety CoC671 showed maximum shoot formation (93.33%) on MS medium supplemented with only Kinetin( 0.5mg/L), 3% sucrose, 10% CW. The best response in terms of shoot multiplication for variety Co86032 was observed on MS medium with BAP (0.2mg/L) and Kinetin (0.1mg/L)with 3% commercial sugar while for variety CoC671 it was observed on MS medium with Kinetin and BAP (0.2mg/L each) and 3.0% commercial sugar. Variety Co86032 produced multiple shoots with an average number of 31.25±0.99 shoots per bottle while variety CoC671 produced 27.72±0.66 shoots per bottle. For rooting, the multiplying shoots were separated in smaller groups and transferred on half strength MS liquid medium containing different concentrations of NAA or IBA with different concentrations of commercial sugar. Half strength MS basal medium with NAA (5.0 mg/L) and 5.0% commercial sugar induced highest rooting (84.44%) with average root number per shoot of 8.49 ±0.25 in Co86032. In variety CoC671, half strength MS basal medium with IBA (5.0 mg/L) and 5.0% commercial sugar induced highest rooting (82.22%) with average root number per shoot of 6.72 ±0.48. The use of commercial sugar can effectively replace the use of expensive sucrose in in vitro multiplication of sugarcane varieties. All culture media were used in liquid state instead of semisolid agar media. Rooted shoots were transplanted in green house for hardening. Use of liquid media, chemical sterilant Sodium hypochlorite (NaOCl) and commercial sugar helped in reducing the cost per plantlet to a considerable extent.

English

The present study deals with the mass propagation of Orthosiphon aristatus - a multipurpose highly valuable ethnomedicinal shrub. Stem and inflorescence explants were inoculated into the Murashige and Skoog medium (MS medium) fortified with different concentrations and combinations of auxins and cytokinins. The maximum number of shoots achieved was 25 &plusmn; 0.78 and 17 &plusmn; 0.67 at 5&nbsp;mg/l benzylaminopurine (BAP) + 2.5 mg/l kinetin (kn) and 3mg/l BAP + 1.5mg/l Kn from nodal and inflorescence explants, respectively. For in vitro rhizogenesis, the elongated micro shoots were aseptically excised and were transferred to the half strength MS liquid medium supplemented with different concentrations of Indole-3 acetic acid and Indole butyric acid (IAA and IBA). The best rooting was achieved at 2 mg/l IBA giving maximum of 12 &plusmn; 0.67 roots per shoot. The well established plantlets were hardened by transferring them to the poly cups containing sterile soil and vermicompost in the ratio of 1:1. After 3 weeks of hardening, established plantlets were transferred to the field with 95% survivability. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Key words: Stem, inflorescence, callus, multiple shoots, acclimatisation, O.aristatus.

Rhizogenesis in Cell Suspension Culture from Mango Ginger: A Source of Isosorbide and n-Hexadecanoic Acid

Curcuma amada Roxb. belongs to the monocotyledonous family Zingiberaceae. It is commonly known as mango ginger and used as a spice and valuable medicine. In this study, adventitious roots of C. amada have been successfully established from cell suspension culture. The highest percentage of adventitious root production was obtained from friable callus derived cell suspension culture. The culture conditions of adventitious root were optimized and the maximum adventitious root production was obtained in half strength MS liquid medium containing 0.3 mg L−1 IBA along with 3% of sucrose after 5 weeks of culture. Among the different initial inoculum density, the best culture condition for root growth occurred at 10 g FW of initial inoculum density. GC-MS analysis revealed that the in vitro raised adventitious roots containing two valuable bioactive compounds, isosorbide and n-hexadecanoic acid. The outcome of the present work will be helpful for the large scale cultivation of adventitious roots for the production of valuable bioactive compounds.

In vitro propagation and withaferin A production in Withania ashwagandha, a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15mg/g of DW) production was obtained on MS medium supplemented with 5.0μM 6-benzyladenine (BA) and 1.0μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90% survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0μM 6-benzyladenine (BA) and 1.0μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.

In vitro development of embryogenic calli and embryogenic stages in suspension cultures of mangosteen (Garcinia mangostana L.)

In this study, the potential of uncoated and coated mangosteen seed explants in forming embryogenic callus were examined in the basal Linsmaier and Skoog medium supplemented with different auxins at various concentrations. Among the highest percentage of callus response (93.3%) was obtained when uncoated seed explants were cultured in basal LS medium containing 8 mg/L 2,4-D. Combining of cytokinins and 2,4-D to improve embryogenicity of calli showed that among the highest percentage of callus response (80%) and the lowest percentage of callus browning (53.53%) with yellowish and compact nodular calli were obtained on MS medum supplemented with 8 mg/L 2,4-D and 0.1 mg/L BAP. Addition of glutamine into MS medium containing 8 and 0.1 mg/L BAP performed that glutamine did not increase the growth of calli, however texture (friable) and color callus (yellowish) was improved. The growth and multiplication of cells in suspension cultures showed that the cells were able to divide and proliferate even though cultured in half strength MS liquid medium without 2,4-D. After six months of culture, the heart embryogenic stage was obtained only on medium supplemented with 1 mg/L BAP. The globular and torpedo embryogenic stages were obtained on media supplemented with 1, 3 and 9 mg/L TDZ after five months of culture. Key words: Mangosteen, embryogenic callus, suspension culture, in vitro.

Improved Clonal Propagation of &amp;lt;i&amp;gt;Alpinia calcarata&amp;lt;/i&amp;gt; Rosc., a Commercially Important Medicinal Plant and Evaluation of Chemical Fidelity through Comparison of Volatile Compounds

An efficient and improved clonal propagation of Alpinia calcarata, a commercially important medicinal plant was established on Murashige and Skoog medium. The axillary shoot proliferation was achieved with maximum 5.2 ± 0.7 shoots in 92.8% of rhizome explants in medium with 2.0 mg/L 6-benzylamiopurine (BAP) and 0.2 mg/L indole-3-acetic acid (IAA). Axillary shoot buds (60%) upon subculture for 8 weeks in the same medium produced multiple shoot initials (12.1 ± 0.4) mediated with meristemoids (4.0 ± 0.5) and callus. A gradual reduction in the concentration of BAP or elimination of IAA was required for rapid induction of normal plants devoid of callus from propagules during subsequent subculture. Single clump of 3-4 multiple shoot initials during second subculture on medium with 1.0 mg/L BAP and 0.1 mg/L IAA yielded an average of 21 shoots which was best among different propagules tried. The shoot multiplication rate was further enhanced to 32 shoots when the similar propagules passed to third subculture on medium with 1.0 mg/L BAP alone. Clumps of multiple shoot initials upon subculture on medium with 1.0 mg/L BAP alone exhibited 10 fold multiplication rates. Use of liquid medium in culture bottles with polypropylene caps supported fast growth of the shoots and spontaneous root formation on 50% of the shoots. Shoots transferred to half-strength MS liquid medium with 0.2 mg/L of IAA and IBA was optimum for maximum roots (8.14 ± 1.34) in 100% shoots. The rooted plants were hardened in mist chamber showed 95% survival and well established in the field. The acclimatized plants showed rhizome formation after 4-6 weeks of growth under shade house. Volatile chemicals profile of the leaves, rhizome and root of the in vitro and conventionally propagated plants analyzed by gas chromatography-mass spectrometry were qualitatively and quantitatively similar. The analysis of growth characteristics of 36 month old in vitro and conventionally propagated plants showed a 50% increment of rhizome fresh biomass with prolific root and leaf growth in the former than the latter ones. The protocol described herein will have practical applications for the large scale production of uniform efficient plants for commercial cultivation of A. calcarata.

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.

Effect of growth regulators on in vitro multiplication and rooting of shoot cultures in sugarcane

An experiment was carried out to study the effect of growth regulators on in vitro multiplication of shoot cultures and their rooting in sugarcane varieties CoS 99259 and CoSe 01235. About 1.5 cm long shoot tip expiants comprising apical meristems and 1–2 leaf primordia were carefully excised from the apical region, sterilized and inoculated on agar (8.0 g/1) gelled MS medium supplemented with various concentrations and combinations of BAP, kinetin and NAA. The results showed that MS medium supplemented with BAP, Kinetin and NAA (0.5 mg/1 each) was most suitable for producing optimum number of shoots in both the varieties. For rooting, the multiplying cultures were separated in smaller groups and transferred on half strength MS liquid medium containing 50g/1 sucrose and different concentrations of auxins (NAA and IBA). Maximum 94% shoot cultures developed vigorous root system on half strength MS liquid medium containing 50 g/1 sucrose and 5.0 mg/1 NAA.

High Frequency Multiple Shoot Induction of Curculigo orchioides Gaertn.: Shoot Tip V/S Rhizome Disc

Curculigo orchioides Gaertn. is an endangered medicinal plant with anticancer properties. The rhizome and tuberous roots of the plant have been used extensively in India in indigenous medicine. Due to its multiple uses, the demand for Curculigo orchioides is constantly on the rise; however, the supply is rather erratic and inadequate. Destructive harvesting, combined with habitat destruction in the form of deforestation has aggravated the problem. The plant is now considered 'endangered' in its natural habitat. Therefore, the need for conservation of this plant is crucial. Here, we describe a successful protocol for multiple shoot induction of C. orchioides using shoot tip and rhizome disc. We find that proximal rhizome discs are optimal for high frequency shoot bud formation than shoot tip and distal rhizome disc. We observed a synergistic effect between 6-benzylaminopurine (BAP) and kinetin (KN) (each at 1 mg/L) on the regeneration of shoot buds from proximal rhizome disc than shoot tip explant. Optimum root induction was achieved on half-strength MS liquid medium supplemented with 1 mg/L of indole-3-butyric acid (IBA). The in vitro raised plantlets were acclimatized in green house and successfully transplanted to natural condition with 90% survival.

In vitro clonal propagation of Mucuna pruriens var. utilis and its evaluation of genetic stability through RAPD markers

The Mucuna pruriens var. utilis is an important legume cover crop. Almost all the parts of the plant are reported to contain L-3,4-dihydroxy phenylalanine (L-Dopa). Here we report a rapid and reliable method for high fidelity micro-propagation. Auxiliary bud explants from 14-day-old seedlings were cultured on Murashige and Skoog’s (MS) medium supplemented with different concentrations of cytokinins. During the first culture on 3.5
M 6-benzylamino purine (BAP) maximum of 6.70 ± 1.15 shoots with an average shoot-length of 1.07 ± 0.21 cm were produced. The number of shoots increased up to 16.33 ± 0.58 recording average length of 1.16 ± 0.29 cm, when the intact shoots were subjected to re-culturing on the same hormonal medium. The shoots exhibited adequate elongation of 4.00 cm on 2.89
M gibberellic acid (GA3). The elongated shoots produced a maximum of 16.67 ± 2.89 roots on half-strength MS liquid medium supplemented with 16.20 iM -naphthalene acetic acid (NAA). The plantlets were acclimatized by transferring them first to peat moss: compost (1:1) mixture followed by sand: soil (1:1) mixture, recording 95% survival. The genetic fidelity of the regenerated shoots was confirmed using randomly amplified polymorphic DNA (RAPD) analysis employing 15 operon primers. This system provides high fidelity micro-propagation system for efficient and rapid micro-propagation of this important green manure cover crop with medicinal properties.

Hyperaccumulation of arsenic by callus, sporophytes and gametophytes of Pteris vittata cultured in vitro

The callus of Pteris vittata was induced from gametophytes generated from spores in vitro, and grew rapidly with periodical medium change. Arsenic tolerance and accumulation of P. vittata callus were compared with those of Arabidopsis thaliana callus. Cell death was not detected in P. vittata callus even at arsenate concentrations up to 2 mM; however, A. thaliana callus died at low (0.2 mM) arsenate concentrations. Meanwhile, P. vittata callus accumulated almost three times more As than A. thaliana callus when exposed to 0.2 mM arsenate. About 60% of the total As was removed when 7.5 g of P. vittata callus was cultured on 150 ml of half-strength MS liquid medium containing 450 microg As for 2 days. Furthermore, P. vittata callus, sporophytes, and gametophytes all grew well under 1 mM of arsenate and accumulated 1,250; 1,150 and 2,180 mg kg(-1 )dry weight As when grown on 2 mM arsenate for 15 or 30 days. The characteristics of non-differentiated cells, large biomass, ease of culture, good synchronization, and excellent As sequestering, make the callus of P. vittata a new ideal system to study the mechanisms of As hyperaccumulation and phytoremediation in As-contaminated groundwater.

Improved in vitro rooting and hyperhydricity in regenerating tissues of Thapsia garganica L.

Stock cultures of Thapsia garganica grown on Murashige and Skoog agar medium (1962) (MS) (0.8% agar [w/v]; pH 5.8) with 0.5 mg l−1 NAA and 1.5 mg l−1 BA were best rooted by subjecting to half strength MS liquid medium with IBA (10 mg l−1) for 3 days prior to transfer to medium without plant growth regulators. A rooting frequency of 60% was noted with seven roots per rooted plant. Rooting was apparent after 10 days. The present study also aimed at reducing the occurrence of hyperhydric plants. The inclusion of 2% polyethylene glycol (w/v) in the growth medium or ventilation of cultures prior to acclimatization resulted in the production of plants true to type, closely resembling wild plants. Those plantlets that had been rooted and exposed to a better vented environment were able to acclimatize readily. Tissue culture propagation is therefore beneficial to the conservation of the medicinally important species, Thapsia garganica.