Solid Murashige And Skoog Medium Research Articles

Tissue culture of Sophora tonkinensis Gapnep. and its quality evaluation

Background:Sophora tonkinensis Gapnep. is an important rare medicinal plant in China. There were only a few papers on the rapid propagation of S. tonkinensis through in vitro tissue culture, and still no report focuses on the quality analysis of in vitro tissue culture plantlets.Materials and Methods:The different concentrations of 6-benzylaminopurine (BAP), kinetin (KT), and indole-3-acetic acid (IAA) were used to establish and screen the optimal rapid propagation technology of S. tonkinensis by orthogonal test; the different concentrations of a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and ABT rooting power (ABT) were used to screen the optimal rooting technology. For quality evaluation of tissue culture plants, three different sites were chose to finish planting experiment. The leaf characteristics, radix ex rhizoma yield, and contents of matrine and oxymatrine were evaluated, respectively, to provide evidence of high yield and good qualities of tissue culture plants.Results:A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog (MS) medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA, and 0.5 mg/l KT; the best root induction medium was solid MS medium at half the macronutrient concentration supplemented with 1.0 mg/l NAA, 0.4 mg/l IBA, and 0.1 mg/l ABT. The rooting rate was 98%. All tissue culture plants showed normal leaf characteristics. Tissue culture plants from two sites possessed higher radix ex rhizoma yield and overall productivity of matrine and oxymatrine than those of seed plants.Conclusion:Tissue culture is a rapid, effective, and convenient propagation method for S. tonkinensis, and the quality of S. tonkinensis tissue culture plants meets the requirement of quality standard of China Pharmacopoeia (edition 2010), the crude drug from S. tonkinensis tissue culture plants will be suitable for substituting the crude drug from seed plants.

Efficient nursery production and multiple-shoot clumps formation from shoot tiller-derived shoot apices of dwarf napiergrass (Pennisetum purpureum Schumach.)

The objective of this study was to improve an in vitro tissue culture system for the dwarf napiergrass l.Pennisetum purpureum Schumach.). Additionally, it was needed special treatments for acclimatization, and carried the risks of somaclonal variation and physiological aberations. Shoot apices as initial explants were isolated aseptically from shoottillers, and cultured on in vitro MS (Murashige and Skoog, 1962) medium containing 3.002 sucrose and 0.3o/o phytagel. The most effective phltohormone treatment for multiple-shoot clumps (MSCs) induction was 0.1 mg L-l 2,4-D plus 2.0 mg L-1 BAP. The addition of 50 p M CuSO+ could increase the percentage of MSCs proliferation. Plant regeneration frequency was achieved up to 84%o by culturing the MSCs on solid MS medium containing 0.1 mg L-1 NAA and2.0 mg L-l BAP. A11 regenerants were successfully grown up in the soil. Compared to control plants, in vitro regefierated plants did not reveal any significant difference (P > 0.05) on morphological characteristics and DNA content. The results of this study suggest that improved protocols for in vitro propagation would provide high quality nursery plant production of dwarf napiergrass in grassland field. Key words: dwarf napiergrass; flow cytometry; multiple-shoot clumps formation; plant regeneration.

Efficient nursery plant production of dwarf cogongrass (Imperata cylindrica L.) through mass propagation in liquid culture

AbstractDwarf cogongrass (Imperata cylindrica L.) was developed as a dwarf mutant through heavy‐ion beam irradiation 7 years ago. The dwarf mutant could be expected to use as a new variety for a cover plant with low maintenance, although it has poor seed fertility. To establish an efficient nursery production system for dwarf cogongrass, we attempted mass propagation of it in liquid culture and investigated the genetic stability of its regenerants. Multiple‐shoot clumps (MSCs) were initiated from apical meristems of dwarf cogongrass on Murashige and Skoog (MS) solid medium supplemented with 0.1 mg L−1 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 2.0 mg L−1 benzylaminopurine (BAP). Mass propagation conditions were established from MSCs cultured in MS liquid medium containing 0.05 mg L−1 2,4‐D and 0.5 mg L−1 BAP. The fresh weight of the MSCs per flask increased by more than 16 times in 14 days of liquid culture. Two different sizes of MSCs were produced in liquid culture. When smaller MSCs (<2 mm in diameter) were transferred to half‐strength hormone‐free MS solid medium, plant regeneration occurred at high frequency (93.3%). These tissues showed high regenerative potential with approximately 350 green shoots recovered within 50 days from 60 regenerating clumps. Furthermore, root elongation was vigorous in the regenerants growing in the same medium. Regenerated plants were acclimatized in hydrated Jiffy‐7 pellets for 30 days and then grown in soil as nursery plants. The plant height of regenerants was almost the same as original dwarf cogongrass and significantly lower than the wild type plants (P < 0.01). Analysis of amplified fragment length polymorphism (AFLP) banding patterns generated using 10 primer combinations showed no major genetic variations among the regenerated plants and original dwarf cogongrass.

The plant activator BTH promotes Ornithogalum dubium and O. thyrsoides differentiation and regeneration in vitro

Benzothiadiazole (BTH) is a structural analogue of salicylic acid (SA) which is widely recognized for its role in elicitation of systemic acquired resistance in a broad range of plant species. Here, BTH was applied to cell cultures of the bulbous ornamental plants Ornithogalum dubium and O. thyrsoides, showing a strong effect on rates of differentiation and morphogenesis. Morphogenic cell clusters in liquid Murashige and Skoog (MS) medium containing 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) were used for all treatments. The calluses were washed thoroughly and activated with increasing concentrations of BTH. Following the induction, calli were grown on a solid MS medium without growth regulators (MS) or on a comparable media with NAA and BAP (M-206). The calli treated with BTH displayed a dose dependent increase in formation of meristematic centres followed by enhanced shoot formation compared to controls. Microscopic analyses revealed increased differentiation to cell organelles and a strengthening of the cell wall. A stronger response to BTH was observed in MS than in M-206 medium. A similar effect on calli differentiation was obtained by three weeks darkness followed by light exposure. The dark/light positive effect on differentiation was further augmented by BTH in a synergistic fashion. It is suggested that BTH enhances the rates of morphogenesis in Ornithogalum cultures by triggering a plant regulator-like activity.

Plant regeneration and stimulation of in vitro flowering in Eruca sativa Mill.

Explants such as apical buds, axillary buds, cotyledons, cotyledonary nodes, leaves, hypocotyls and immature embryonal axes from in vitro -grown plantlets were inoculated on the Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzylaminopurine in combination with 2.85 μM indole-3-acetic acid. Best multiple shoots formation was obtained with cotyledonary nodes. Each inoculated explant produced 18.10 ± 0.66 shoots within 2 to 3 weeks. These shoots were separated carefully and were transferred to the fresh half strength MS solid medium with indole-3-butyric acid (4.90 μM) for the development of the roots. These in vitro -developed plantlets produced flowers on the same medium with supplementation of 6-furfuril kinetin (0.23 μM). These plantlets were successfully transferred to the soil where they grew well for 8 to 10 weeks with 80% survivability. Keywords: Eruca sativa , cotyledonary nodes, in vitro regeneration, in vitro flowering, shoot multiplication

Production of plants from callus of Vigna radiata stem seedlings

The present study include callus initiation from leaves and stems explants of Vigna radiata seedlings by using solidified Murashige and Skoog (MS) medium supplemented with different concentrations of Naphthalene acetic acide NAA (0.5,1.0,2.0)mg/L and benzyl adenin BA (0.1,0.2,0.3,0.5,1.0,2.0)mg/L. The results showed that Ms medium which contains 2.0 mg/l of NAA was the best one for callus initiation from leaves while solidified MS medium containing 0.5 mg/L NAA and 2.0 mg/L BA was superior for callus induction from stem. The callus produced from stems and leaves has been maintained on solidified MS medium supplemented with 0.2 mg/L Kin and 0.2 mg/L 2,4-D for shoot regeneration, MS media containing 0.5mg/L NAA and different concentration of BA (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0)mg/L were used. The results indicated that MS medium with 5.0 mg/L BA was the best one for shoot regeneration. Moreover, ther was a difficulty in rooting the regenerated shoots, Since they failed to produce roots in the rooting media which were used except solidified MS medium supplemented with 2mg/L NAA.

Cytotoxic activity of ethanol extracts of in vitro grown Cistus creticus subsp. creticus L. on human cancer cell lines

Cistus creticus subsp. creticus is a native plant of the Mediterranean region and it has been used since ancient times for its medicinal properties. The leaves secrete labdane type diterpenes that exhibit considerable antibacterial and cytotoxic activities. However there are no data about implementation of in vitro culture of C. creticus in order to obtain applicable production of labdane diterpenes. We successfully established in vitro culture of shoots and roots on solid or liquid MS (Murashige and Skoog) medium without addition of plant growth regulators. GC/FID and GC/MS detections were used to carry out characterization of ethanol extracts of shoots and roots from in vitro culture. Labdane diterpenes were the most abundant compounds in shoot extract, but absent from the root extract. Total phenolic and flavonoid contents of C. creticus shoot and root ethanol extracts were also determined. In order to evaluate possible cytotoxic activity of extracts, sulforhodamine B (SRB) assay was performed on five human cancer cell lines. Only shoot extract had cytotoxic activity on HeLa (cervix), MDA-MB-453 (breast) and FemX (melanoma) cancer cells with IC50 reaching 80.83μg/ml (HeLa), 76.18μg/ml (MDA-MB-453), and 87.52μg/ml (FemX) respectively. This is the first report of cytotoxic effect of labdane diterpenes from Cistus on HeLa cells.

Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (Sorbus pohuashanensis)

Plant regeneration through somatic embryogenesis was achieved using immature zygotic embryos (ZE) of Sorbus pohuashanensis as explants. Over 50% of immature ZEs from immature seed collected at 30 days after pollination produced direct somatic embryos (SEs) on Murashige and Skoog (MS) medium supplemented with 0–0.44 μM 6-benzyladenine (BA) in combination with 5.73 μM naphthaleneacetic acid (NAA) or with 0.91–2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Fourteen to 23 SEs per explant were regenerated on MS medium supplemented with BA 0.44 μM in combination with NAA 5.73 μM. SE formation decreased when sucrose concentrations were higher than 40 g L−1. Repetitive embryogenesis occurred following culture on solid MS medium containing 12 μM abscisic acid, 75 g L−1 polyethylene glycol, and 20 g L−1 sucrose at 25 ± 1°C under a 16-h photoperiod with a light intensity of 40 μmol m−2 s−1. Over 40% of the mature SEs germinated on solid MS medium under light condition described previously. Up to 40% of the regenerated plantlets were successfully acclimatized under greenhouse conditions. Plantlets derived from SEs grew vigorously with similar morphology as those germinated from ZEs. Histological studies of explants at various developmental stages of somatic embryogenesis revealed that SEs passed through globular, heart, torpedo, and mature stages. Similar to ZE suspensors, similar structures of SE degenerated in later stages of embryo development. ZE and SE are a effective means of regenerating tissue culture plantlets for S. pohuashanesis.

Luteolin content and antioxidant activity in micropropagated plants of Poliomintha glabrescens (Gray)

Mexican oregano is composed by a wide range of endemic species which support important economic activities, but with serious limitations. In this work a micropropagation protocol was established to regenerate oregano plants (Poliomintha glabrescens Gray) from apical buds. At the same time we evaluated the impact of this process in the phytochemical profile of the new plants. The optimal induction of apical buds was observed when shoot-tip explants were incubated on solidified Murashige and Skoog (MS) medium supplemented with 0.5 mg ml−1 α-naphthalenacetic acid (NAA) and 0.02 mg ml−1 6-benzyladenine. Also, the optimal response to root induction was with MS solid medium added with 0.5 mg ml−1 of NAA. The micropropagation protocol took 17 weeks with an efficiency of 40%, and plants achieved were successfully acclimatized under greenhouse conditions. The total phenolics (TP) concentration, antioxidant capacity (oxygen radical absorbance capacity, ORAC), and luteolin content from methanol extracts of wild type (WT) and in vitro (IN) and ex vitro (EX) plants were determined. The results show that TP content and ORAC were similar for WT and EX plants, but IN plants had the highest TP content and ORAC activity (25 and 28% respectively). Luteolin content showed significant differences between WT, IN and EX, with EX plants having the highest luteolin content (10% more compared to WT). In summary, we successfully microprogated oregano plants from WT and they contained similar amount of their TP content and ORAC activity and an increase in luteolin content.

Establishment of Cell Suspension Cultures and Plant Regeneration in White Dandelion (Taraxacum coreanum NAKAI.)

In this study, we established a novel somatic embryogenesis and plant regeneration system through cell suspension culture of white dandelion (Taraxacum coreanum NAKAI.). Embryogenic calli could be initiated from leaf and root explants of sterile seedlings on solid Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 3-week cultures. To proliferate embryogenic calli rapidly, cell suspension culture was performed with transferred to liquid MS medium with various combinations of plant growth regulators (PGRs) including 2,4-D, <TEX>${\alpha}$</TEX>-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), <TEX>$N^6$</TEX>-benzylamino purine (BAP), thidiazuron (TDZ), and kinetin. During suspension cultures, embryogenic calli not only greatly proliferated, but shoot organogenesis also simultaneously occurred from the surface of somatic embryos. Among them, TDZ at lower concentration, 0.1 mg/L produced the highest efficiency of somatic embryo formation and shoot organogenesis. Rooting of embryogenic calli with adventitious shoots was done on solid MS medium containing 0.1 mg/L NAA and 0.3% activated carbon. Nearly 80% of embryogenic calli with shoot organogenesis could be rooted normal. Well-rooted plantlets were transferred into pots under a greenhouse condition, and plants derived from this system appeared phenotypically normal.

Induction of anthocyanin pigments in callus cultures of Rosa hybrida L. in response to sucrose and ammonical nitrogen levels

The influence of varied concentrations of sucrose and ammonical (NH4+) nitrogen on in vitro induction and expression of anthocyanin pigments from Rosa hybrida cv. ‘Pusa Ajay’ was investigated. Of two explants (petal and leaf discs) selected and cultured under two different conditions (light and dark), leaf discs were found to be most suitable for callus initiation. Profuse and early callus induction was observed when leaf discs of rose were cultured under total dark conditions on solid Murashige and Skoog (MS) medium supplemented with 4.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). Early pigment initiation, enhancement and maximum anthocyanin production from calluses were recorded when leaf discs were cultured on Euphorbia millii (EM) medium supplemented with 7% sucrose compared with calluses cultured at 4% sucrose concentration under 16/8 h (light/dark) photoperiod regime. Reducing the concentration of NH4+ nitrogen in the solid MS medium led to slight improvement in anthocyanin production in rose leaf calluses.

Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m−2 s−1, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.

In Vitro Regeneration of Cephalotus follicularis

To establish a mass micropropagation procedure for Cephalotus follicularis, the effects of varying the strengths of solid Murashige and Skoog (MS) medium were investigated using subcultured shoot explants. After a 60-day primary culture from root mass, the regenerated shoot explants were subcultured every 60 days in solid MS medium. To facilitate shoot proliferation, liquid MS medium was applied with or without exogenous auxin and cytokinin. Our results demonstrate that shoot proliferation and survival of C. follicularis is most effective in modified MS (MMS) medium containing one-fifth or one-tenth strength macronutrients and full-strength micronutrients. Successful shoot proliferation and development of C. follicularis explants were obtained in one-fifth or one-tenth modified liquid MS medium without auxin and cytokinin or with addition of 5 μM indole 3-acetic acid/1 μM N6-benzyladenine for 45 days. The liquid medium consistently produced more explants than the solid medium and shortened the culturing time. Plantlets cultured in hormone-free one-fifth MMS medium developed greater root systems. Using the liquid culture we established, vigorous plants with extensive roots were obtained within 4 months. Plant survival in the greenhouse reached 100%.

Use of protocorm-like bodies for studying alkaloid metabolism in Pinellia ternata

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant Emmenopterys henryi Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS2) for 30 min at 0°C and followed by 100% PVS2 for 40 min at 0°C. After changing the solution with fresh 100% PVS2, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l−1, α-naphthaleneacetic acid 0.1 mg l−1, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.

Plant regeneration in Robinia pseudoacacia from cell suspension cultures

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1-3 × 104 cells per cm3 of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm-3 benzyladenine (BA) along with 0.05 mg dm-3 &alpha;-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm-3 BA. These microshoots after dipping in 1-2 cm3 of 10 mg dm-3 indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80-82 % rooting within 4 weeks.

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L−1 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA3) at 1.0 μmol L−1 stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L−1 of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×105 protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×105−6×105 protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.

PROTOCORM-LIKE BODY FORMATION AND PLANT REGENERATION OF DENDROBIUM HUOSHANENSE, AN ENDANGERED MEDICINAL PLANT

A procedure was developed for protocorm-like bodies (PLBs) formation and proliferation and subsequently plant regeneration from stem segments of Dendrobium huoshanense Tang et Cheng, an endangered medicinal plan. PLBs were formed on Murashige and Skoog (MS) medium containing α-naphthaleneacetic acid (NAA) alone or in combination with kinetin (KIN). The highest frequency (79.9%) of PLB formation occurred on MS medium supplemented with 7.5 μmol L -1 NAA and 0.5 μmol L -1 KIN. Growth regulator-free MS medium at half strength was optimum for PLB proliferation and liquid suspension culture gave the maximum increase in fresh weight after culture of 4 weeks in the dark. Upon transfer to solid MS medium without growth regulators in the light, the proliferated PLBs developed into shoots. Potato extract enhanced the regeneration and elongation of shoots and approximately 420 shoots were obtained from one gram PLBs. High frequency of shoots (90.7%) were rooted on MS medium supplemented with 10% banana extract and complete plantlet with 3-5 roots were obtained after 7 weeks of rooting initiation.

In vitro Organogenesis and Alkaloid Accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.